Method and pharmaceutical composition for treating inflammation

ABSTRACT

A method of treating an inflammation in a subject in need thereof is disclosed. The method comprises locally or systemically administering to the subject IFN-gamma in an amount so as to achieve an IFN-gamma bulk tissue concentration at a site of inflammation of 1-8,000 units per kilogram body weight, thereby ameliorating the inflammation.

RELATED APPLICATIONS

This is a Divisional of U.S. patent application Ser. No. 11/090,188filed Sep. 17, 2001 which is a continuation of U.S. patent applicationSer. No. 09/953,206, now U.S. Pat. No. 6,911,198 issued on Jun. 28,2005.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates in general to methods and pharmaceuticalcompositions for treating inflammation, and more particularly, to amethod and pharmaceutical composition for downregulating adhesion andmigration of lymphocytes and, specifically, to the administration ofultra-low dosages of interferon gamma (IFN-γ) to treat a variety ofimmunopathological states which are accompanied by inflammation, suchas, but not limited to, autoimmune disease, allergy, inflammation andgraft rejection.

Many lymphocyte-mediated immune processes require adhesion oflymphocytes to the ECM. Such adhesion is critical, for example, for Band T lymphocyte extravasation and migration during differentiation,activation, proliferation and effector function during both normal andpathological immune processes. As such, selectively interfering withadhesion and migration of lymphocytes can be directly and effectivelyapplied towards treatment of disorders involving lymphocyte-mediatedpathogenesis.

Specific Immunity:

The specific arm of the immune system employs B and T lymphocytes torecognize and eliminate foreign antigens, such as foreignmicroorganisms, foreign substances or allogeneic cells, while remainingunresponsive to self antigens. Targeting of specific foreign antigensand elimination of foreign bodies displaying these is mediated via B andT lymphocytes which, following somatic gene rearrangement and clonalselection, respectively express antibodies specific to a particularforeign antigen or T cell receptors (TCRs) specific to a particularmajor histocompatibility complex (MHC)-foreign peptide complex. Thehumoral arm of the immune system thus employs antibodies to eliminate,for example, bacteria and foreign substances, whereas the cellular armof the immune system employs T lymphocytes to eliminate, for example,virus- or parasite-infected cells, cells expressing mutatedself-antigens, such as tumor cells or allogeneic cells.

Trafficking of Lymphocytes During Immune Responses

During the processes of immune surveillance and specific immuneresponses, lymphocytes extravasate and migrate to secondary lymphoidorgans, such as lymph nodes (LNs), for antigen-specific activation afterwhich these migrate to the sites of inflammation for execution ofeffector functions against foreign pathogens. Extravasation oflymphocytes to such sites occurs through specialized high endothelialvenules (HEV) via recognition of organ-specific adhesion molecules bycounter-receptors, such as integrins, expressed on lymphocytes (Bradley,L., and Watson, S. R. Curr. Opin. Immunol. 1996, 8: 312; Imhof, B. A.,and Dunon, D. Adv. Immunol. 1995, 58: 345). These processes involvemultiple adhesion steps regulated via a combinatorial series ofmolecular events.

Continuous recirculation of lymphocytes to organized lymphoid tissue orto sites of antigen-presentation, such as LNs, is necessary for thedevelopment of primary immune responses to foreign antigen bylymphocytes and such recirculation from one anatomic compartment toanother is critically dependent on adhesive interactions of lymphocyteswith endothelium and with the extracellular matrix (ECM) forextravasation and migration within tissues, such as sites ofinflammation, respectively.

The process of B cell development also involves distinct traffickingpatterns and thus is also critically dependent on regulation of adhesionof B lymphocytes to endothelium and to ECM. Immature B cells initiallydifferentiate in the bone marrow from which they exit to the peripheryand selectively migrate into the spleen to complete their maturation.While in transit to the spleen, specific mechanisms prevent the entryinto and/or retention of immature B cells in sites ofantigen-presentation, such as sites of inflammation or secondarylymphoid organs, such as LNs. This is due to the fact that maturation ofB cells occurs in anatomic compartments in which auto-antigens inducethe clonal deletion of autoreactive immature B cells. Thus, such cellsare prevented from entering sites of antigen-presentation so as toprevent deletion of foreign antigen-reactive clones.

The specific mechanisms of lymphocyte trafficking during immuneresponses are best characterized for T cells, as detailed below.

Non-activated T lymphocytes traffic through the T cell areas ofsecondary lymphoid organs, such as LNs, where they encounter antigenpresented by dendritic cells (DCs), thereby triggering activation ofantigen-specific T cells (Butcher, E. C., and Picker, L. J. Science1996, 272: 60; Banchereau, J., and Steinman, R. M. Nature 1998, 392:245). In the presence of polarizing cytokines, activated T lymphocytesacquire effector functions and differentiate into Th1 or Th2 subtypesdisplaying characteristic cytokine production profiles and mediatingpro-inflammatory and allergic types of responses, respectively (Abbas,A. K. et al., Nature 1996, 383: 787; O'Garra, A. Immunity 1998, 8: 275).Such differentiated T cells downregulate LN homing receptors andupregulate specific tissue homing receptors (Xie, H. et al., J. Exp.Med. 1999, 189: 1765; Potsch, C. et al., Eur. J. Immunol. 1999, 29:3562; Campbell, J. J., and Butcher, E. C. Curr. Opin. Immunol. 2000, 12:336; Sallusto, F. et al., Annu. Rev. Immunol. 2000, 18: 593) resultingin Th1 and Th2 cells exhibiting distinct migratory profiles in vivo(Austrup, F. et al., Nature 1997, 385: 81; Randolph, D. A. et al.,Science 1999, 286: 2159).

During immune responses to pathogenic insult, foreign antigens presentin affected tissues are taken up by antigen-presenting DCs which migrateto sites of antigen-presentation, such as LNs. Meanwhile, components ofthe non-specific cellular immune system, such as neutrophils and othergranulocytes, initiate inflammation by, for example, releasingpro-inflammatory molecules, such as chemokines and cytokines whichactivate local endothelial cells to upregulate expression oflymphocyte-specific adhesion molecules, such as immunoglobulinsuperfamily ligands. In sites of antigen-presentation, such as LNs, DCspresenting foreign antigens activate antigen-specific lymphocytes.Activated lymphocytes then upregulate adhesion molecules, such asintegrins specific for activated endothelial cells (ECs), exit to thecirculation and extravasate at sites of inflammation.

In the circulation, transient interactions between lymphocytes andendothelium enable lymphocyte tethering and rolling along theendothelial wall. During this rolling phase, lymphocytes are activatedby intracellular signals generated by engaged adhesion molecules, suchas selectins and chemokine receptors, which transduce signals promotingfirm adhesion between integrin molecules, expressed on lymphocytes, andtheir immunoglobulin superfamily ligands expressed on the endothelialwall. Such adherent lymphocytes then extravasate through theintercellular margins of the endothelium and migrate, viaadhesion-disruption cycles, through the ECM to reach foreign-antigencontaining inflamed tissue wherein effector functions are performed(Butcher, E. C. Cell 1991, 67: 1033; Shimizu, Y., W. et al., Immunol.Today 1992, 13: 106; Mackay, C. R., and B. A. Imhof. Immunol. Today1993, 14: 99; Schall, T. J., and K. B. Bacon. Curr. Opin. Immunol. 1994,6: 865; Hogg, N., and C. Berlin. Immunol. Today 1995, 16: 327; Imhof, B.A., and D. Dunon. Adv. Immunol. 1995, 58: 345; Butcher, E. C., and J.Picker. Science 1996, 272: 60; Springer, T. Annu. Rev. Physiol. 1995,57: 827).

The trafficking signals directing activated effector T cells toperipheral tissues are organ-specific and are distinct for the differentsubgroups of T cells. For example, naïve T cells express CD62 ligand(CD62L) and CC chemokine receptor 7 (CCR7), which are required for HEVextravasation (Springer, T. A. Cell 1994, 76: 301; Cyster, J. G. Science1999, 286: 2098; Stein, J. V. et al., J. Exp. Med. 2000, 191: 61;Forster, R. et al., Cell 1999, 99: 23). Upon foreign antigen encounterat sites of inflammation, activated lymphocytes release cytokines andchemokines which amplify the inflammatory cascade thereby increasingvascular and ECM permeability thus facilitating infiltration ofeffectors to inflamed sites of foreign pathogen infiltration. Suchinflammatory processes normally result in a certain amount of tissuedestruction which, when appropriately regulated, is tolerated by thebody as a reversible consequence of optimal immune responses.

Thus, adhesion of lymphocytes to endothelium and the ECM is critical toextravasation and migration during processes such as lymphocytematuration, antigen-specific activation and effector responses againstforeign antigens at sites of inflammation.

Lymphocyte-Mediated Disorders:

Although lymphocyte-mediated immune processes normally serve to allowthe immune system to fight foreign pathogens, in certain contexts suchimmune processes can lead to pathological states referred to ashypersensitivity diseases. Alternatively, transplantation of foreigncells, tissues or organs or surgically implanted prosthetic devices canalso elicit undesirable immune responses against such implants inrecipients subjected to such therapeutic interventions. In suchdiseases, lymphocytic infiltration into tissues and the consequencesthereof, such as cytokine production, significantly contribute toundesirable sequelae, such as acute tissue injury, resulting fromuncontrolled inflammation.

Such uncontrolled inflammation may be a result of antibody-mediateddiseases such as allergy (immediate hypersensitivity) or immune complexdeposition. Alternatively, uncontrolled inflammation may be a result ofT lymphocyte-mediated diseases such as contact dermatitis and drugeruptions (delayed hypersensitivity). Infection or cancer may alsoresult in uncontrolled inflammation.

Importantly, deregulated immune responses may specifically targetself-antigens, either idiopathically, as a result of cross-reactivitywith foreign antigen or, alternately, immunity directed against foreignantigens may cause damage to specific tissues to which such foreignantigens have a specific affinity.

Such hypersensitivity diseases have been implicated in an extremelybroad range of autoimmune diseases including systemic, cutaneous,rheumatoid, cardiovascular, gastrointestinal, hepatic, reproductive,glandular, neurological, muscular, nephric and connective tissueautoimmune diseases, as described in the Preferred Embodiments section.

Role of Lymphocytes in Transplantation Failure:

Immune reactions may also be deleterious in the context of therapeutictissue transplantation. For example, T lymphocytes are responsible fortransplantation-related immunopathologies such as allograft rejection(Krensky A. et al., N Engl J Med. Feb. 22, 1990; 322 (8): 510) and GVHD(Theobald, M. Transfus Sci September 1994; 15 (3): 189) which are themajor causes of transplantation failure. In allograft rejection and inGVHD, respectively, immune responses are mediated by T cells of the hostor of the donor, respectively, which extravasate and migrate into donorand host tissues, respectively, to induce disease. In the case of graftrejection, host T lymphocytes also migrate into secondary lymphoidorgans where they are primed against graft-derived allogeneic antigens.

Prior Art Therapy Using IFN-γ:

Satisfactory IFN-γ-based therapies for many diseases involvinginflammation, such as autoimmune diseases, allergic diseases,transplantation failure and cancer, as described above, have yet to bedeveloped. Prior art approaches of treating inflammation-relateddisorders using IFN-γ have employed administration of high levels ofthis cytokine, as described below.

Animal studies: The use of high levels of IFN-γ to regulate inflammatoryprocesses has been attempted in animal models of autoimmune disease. Ina murine asthma model, treatment of tracheal eosinophil and CD4⁺ T cellinfiltration was achieved using intraperitoneal administration of highlevels of IFN-γ, at doses of 24,000 and 240,000 units per kilogram bodyweight administered per day, Med. 1993, 177: 573).

Human studies: Since high levels of IFN-γ were shown to be of potentialtherapeutic benefit in animal studies, such as the one described above,therapy employing high levels of IFN-γ has been attempted in humanclinical trials for treatment of autoimmune disease. Such treatments,however, have been found to produce unacceptably severe side-effects inpatients.

For example, in a clinical trial of long-term treatment of theinflammatory disease idiopathic pulmonary fibrosis, high levels of IFN-γ(33,000 units per kilogram body weight administered three times perweek) were required in order to provide substantial therapeutic effect(Ziesche, R. et al., N. Engl. J. Med. 1999, 341: 1264). All patientstreated in this study, however, developed fever and chills and one-thirdexperienced significant bone and muscle pain as a result of high-doseIFN-γ administration.

In an open pilot study employing IFN-y therapy for treatment of anotherinflammatory disease, Crohn's disease, administration of high levels ofIFN-γ (15,000 units/kg administered three times per week) led to asubstantial decrease in Crohn's disease activity index in most patients,however, most patients did not finish the 12-week treatment course dueto suboptimized dosages and an unacceptable incidence of side-effects(Debinski H. et al., Ital J Gastroenterol Hepatol October 1997; 29(5):403). It should be noted that although the title of the latterpublication refers to the employed dose of 15,000 units per kilogrambody weight IFN-γ as being a “low” dose, the method of the presentinvention, as described in the Examples section, below, employsultra-low doses of IFN-γ being at least two orders of magnitude lowerthan such doses.

The undesirable side-effects of high doses of IFN-γ were alsodemonstrated in a human clinical trial for treatment of metastatic renalcell carcinoma in which patients were treated with high levels of IFN-γ(10⁷ units IFN-γ per m² per day for 5 days, administered every two weeksfor four weeks, followed by repeated administration of such dose, threetimes a week for 2 weeks. A dose of 10⁷ unitS/m² corresponds to over100,000 units per kg body weight in an average 60 kg human) (Griebel, P.et al., Int. Immunol. 1999, 11: 1139). These patients experienced majorside-effects including fever and chills (75%), anorexia and fatigue(75%), nausea and vomiting (80%), leukopenia (3.8%) and abnormal liverfunction (25%).

Thus, as a result of the significant side-effects of high-dose IFN-γtreatment described above, this cytokine is not currently employedtherapeutically. The deleterious effects of high-dose IFN-γ may resultfrom its potentiation of the effects of other pro-inflammatory mediatorsand upregulation of surface expression of MHC class II in monocytes,macrophages and dendritic cells, as well as of adhesion molecules inendothelial and epithelial cells (Barnes, P. J., and Lim, S. Mol. Med.Today 1998, 4: 452).

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a method of treatment of disease with IFN-γ devoidof the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of treating an inflammation in a subject in need thereof, themethod comprising locally or systemically administering to the subjectIFN-γ in an amount so as to achieve an IFN-γ bulk tissue concentrationat a site of inflammation of 1-8,000 units per kilogram body weight,thereby ameliorating the inflammation.

According to further features in preferred embodiments of the inventiondescribed below, the bulk tissue concentration is 0.1-4,000 units perkilogram body weight.

According to still further features in the described preferredembodiments, the bulk tissue concentration is 0.2-2,000 units perkilogram body weight.

According to still further features in the described preferredembodiments, the bulk tissue concentration is 0.5-1,000 units perkilogram body weight.

According to still further features in the described preferredembodiments, the bulk tissue concentration is 1-500 units per kilogrambody weight.

According to still further features in the described preferredembodiments, the bulk tissue concentration is 2-240 units per kilogrambody weight.

According to still further features in the described preferredembodiments, the bulk tissue concentration is 4-240 units per kilogrambody weight.

According to still further features in the described preferredembodiments, the concentration is sufficiently low so as to avoidunwanted side-effects associated with IFN-γ administration.

According to still further features in the described preferredembodiments, the unwanted side-effects are selected from the groupconsisting of fever, chills, flu symptoms, bone pain, muscle pain,anorexia, fatigue, nausea, vomiting, leukopenia, diarrhea, fatigue,abnormal liver function, black, tarry stools; blood in urine, blood instools, confusion, cough, hoarseness, loss of balance control, mask-likeface, painful urination, difficult urination, pinpoint red spots onskin, shuffling walk, stiffness of arms, stiffness of legs, trembling ofhands, shaking of hands, trembling of fingers, shaking of fingers,trouble in speaking, trouble in swallowing, trouble in thinking, troublein concentrating, trouble in walking, unusual bleeding, unusualbruising, general feeling of discomfort, general feeling of illness,headache, skin rash, unusual tiredness, back pain, side-pain, dizziness,joint pain, loss of appetite and weight loss.

According to still further features in the described preferredembodiments, the inflammation is associated with an inflammatorydisease, disorder or condition.

According to still further features in the described preferredembodiments, the inflammatory disease is selected from the groupconsisting of chronic inflammatory disease and acute inflammatorydisease.

According to still further features in the described preferredembodiments, the inflammation is associated with hypersensitivity.

According to still further features in the described preferredembodiments, the hypersensitivity is selected from the group consistingof Type I hypersensitivity, Type II hypersensitivity, Type IIIhypersensitivity, Type IV hypersensitivity, immediate hypersensitivity,antibody mediated hypersensitivity, immune complex mediatedhypersensitivity, T lymphocyte mediated hypersensitivity and delayedtype hypersensitivity.

According to still further features in the described preferredembodiments, the delayed type hypersensitivity is selected from thegroup consisting of contact dermatitis and drug eruption.

According to still further features in the described preferredembodiments, the T lymphocyte-mediated hypersensitivity is selected fromthe group consisting of helper T lymphocyte mediated hypersensitivityand cytotoxic T lymphocyte mediated hypersensitivity.

According to still further features in the described preferredembodiments, the helper T lymphocyte-mediated hypersensitivity isselected from the group consisting of T_(h)1 lymphocyte mediatedhypersensitivity and T_(h)2 lymphocyte mediated hypersensitivity.

According to still further features in the described preferredembodiments, the inflammation is associated with autoimmune disease.

According to still further features in the described preferredembodiments, the autoimmune disease is selected from the groupconsisting of cardiovascular disease, rheumatoid disease, glandulardisease, gastrointestinal disease, cutaneous disease, hepatic disease,neurological disease, muscular disease, nephric disease, disease relatedto reproduction, connective tissue disease and systemic disease.

According to still further features in the described preferredembodiments, the cardiovascular disease is selected from the groupconsisting of occlusive disease, atherosclerosis, myocardial infarction,thrombosis, Wegener's granulomatosis, Takayasu's arteritis, Kawasakisyndrome, anti-factor VIII autoimmune disease, necrotizing small vesselvasculitis, microscopic polyangiitis, Churg and Strauss syndrome,pauci-immune focal necrotizing glomerulonephritis, crescenticglomerulonephritis, antiphospholipid syndrome, antibody induced heartfailure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiacautoimmunity in Chagas' disease and anti-helper T lymphocyteautoimmunity.

According to still further features in the described preferredembodiments, the rheumatoid disease is selected from the groupconsisting of rheumatoid arthritis and ankylosing spondylitis.

According to still further features in the described preferredembodiments, the glandular disease is selected from the group consistingof pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome.

According to still further features in the described preferredembodiments, the gastrointestinal disease is selected from the groupconsisting of colitis, ileitis, Crohn's disease, chronic inflammatoryintestinal disease and celiac disease.

According to still further features in the described preferredembodiments, the cutaneous disease is selected from the group consistingof autoimmune bullous skin disease, pemphigus vulgaris, bullouspemphigoid and pemphigus foliaceus.

According to still further features in the described preferredembodiments, the hepatic disease is selected from the group consistingof autoimmune hepatitis and primary biliary cirrhosis.

According to still further features in the described preferredembodiments, the neurological disease is selected from the groupconsisting of neurodegenerative disease, multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, myasthenia gravis, motor neuropathy,Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenicsyndrome, paraneoplastic neurological disease, paraneoplastic cerebellaratrophy, non-paraneoplastic stiff man syndrome, progressive cerebellaratrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis,Sydeham chorea, Gilles de la Tourette syndrome, autoimmunepolyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia,arthrogryposis multiplex, optic neuritis and stiff-man syndrome.

According to still further features in the described preferredembodiments, the muscular disease is selected from the group consistingof autoimmune myositis, primary Sjogren's syndrome and smooth muscleautoimmune disease.

According to still further features in the described preferredembodiments, the nephric disease is autoimmune interstitial nephritis.

According to still further features in the described preferredembodiments, the disease related to reproduction is repeated fetal loss.

According to still further features in the described preferredembodiments, the connective tissue disease is selected from the groupconsisting of autoimmune ear disease and autoimmune disease of the innerear.

According to still further features in the described preferredembodiments, the systemic disease is selected from the group consistingof systemic lupus erythematosus and systemic sclerosis.

According to still further features in the described preferredembodiments, the inflammation is associated with an infectious disease.

According to still further features in the described preferredembodiments, the infectious disease is selected from the groupconsisting of chronic infectious disease, subacute infectious disease,acute infectious disease, viral disease, bacterial disease, protozoandisease, parasitic disease, fungal disease, mycoplasma disease and priondisease.

According to still further features in the described preferredembodiments, the inflammation is associated with a disease associatedwith transplantation of a graft.

According to still further features in the described preferredembodiments, the disease is selected from the group consisting of graftrejection, chronic graft rejection, subacute graft rejection, hyperacutegraft rejection, acute graft rejection and graft versus host disease.

According to still further features in the described preferredembodiments, the graft is selected from the group consisting of asyngeneic graft, an allograft and a xenograft.

According to still further features in the described preferredembodiments, the graft is selected from the group consisting of acellular graft, a tissue graft, an organ graft and an appendage graft.

According to still further features in the described preferredembodiments, the cellular graft is selected from the group consisting ofa stem cell graft, a progenitor cell graft, a hematopoietic cell graft,an embryonic cell graft and a nerve cell graft.

According to still further features in the described preferredembodiments, the tissue graft is selected from the group consisting of askin graft, a bone graft, a nerve graft, an intestine graft, a cornealgraft, a cartilage graft, a cardiac tissue graft, a cardiac valve graft,a dental graft, a hair follicle graft and a muscle graft.

According to still further features in the described preferredembodiments, the organ graft is selected from the group consisting of akidney graft, a heart graft, a skin graft, a liver graft, a pancreaticgraft, a lung graft and an intestine graft.

According to still further features in the described preferredembodiments, the appendage graft is selected from the group consistingof an arm graft, a leg graft, a hand graft, a foot graft, a fingergraft, a toe graft and a sexual organ graft.

According to still further features in the described preferredembodiments, the inflammation is associated with an allergic disease.

According to still further features in the described preferredembodiments, the allergic disease is selected from the group consistingof asthma, hives, urticaria, pollen allergy, dust mite allergy, venomallergy, cosmetics allergy, latex allergy, chemical allergy, drugallergy, insect bite allergy, animal dander allergy, stinging plantallergy, poison ivy allergy and food allergy.

According to still further features in the described preferredembodiments, the inflammation is associated with a neurodegenerativedisease.

According to still further features in the described preferredembodiments, the inflammation is associated with a cardiovasculardisease.

According to still further features in the described preferredembodiments, the inflammation is associated with a gastrointestinaldisease.

According to still further features in the described preferredembodiments, the inflammation is associated with a tumor.

According to still further features in the described preferredembodiments, the tumor is selected from the group consisting of amalignant tumor, a benign tumor, a solid tumor, a metastatic tumor and anon-solid tumor.

According to still further features in the described preferredembodiments, the inflammation is associated with septic shock.

According to still further features in the described preferredembodiments, the inflammation is associated with anaphylactic shock.

According to still further features in the described preferredembodiments, the inflammation is associated with toxic shock syndrome.

According to still further features in the described preferredembodiments, the inflammation is associated with cachexia.

According to still further features in the described preferredembodiments, the inflammation is associated with necrosis.

According to still further features in the described preferredembodiments, the inflammation is associated with gangrene.

According to still further features in the described preferredembodiments, the inflammation is associated with a prosthetic implant.

According to still further features in the described preferredembodiments, the prosthetic implant is selected from the groupconsisting of a breast implant, a silicone implant, a dental implant, apenile implant, a cardiac implant, an artificial joint, a bone fracturerepair device, a bone replacement implant, a drug delivery implant, acatheter, a pacemaker and a respirator tube.

According to still further features in the described preferredembodiments, the inflammation is associated with menstruation.

According to still further features in the described preferredembodiments, the inflammation is associated with an ulcer.

According to still further features in the described preferredembodiments, the ulcer is selected from the group consisting of a skinulcer, a bed sore, a gastric ulcer, a peptic ulcer, a buccal ulcer, anasopharyngeal ulcer, an esophageal ulcer, a duodenal ulcer and agastrointestinal ulcer.

According to still further features in the described preferredembodiments, the inflammation is associated with an injury.

According to still further features in the described preferredembodiments, the injury is selected from the group consisting of anabrasion, a bruise, a cut, a puncture wound, a laceration, an impactwound, a concussion, a contusion, a thermal burn, frostbite, a chemicalburn, a sunburn, a desiccation, a radiation burn, a radioactivity burn,smoke inhalation, a torn muscle, a pulled muscle, a torn tendon, apulled tendon, a pulled ligament, a torn ligament, a hyperextension, atorn cartilage, a bone fracture, a pinched nerve and a gunshot wound.

According to still further features in the described preferredembodiments, the inflammation is a musculo-skeletal inflammation.

According to still further features in the described preferredembodiments, the musculo-skeletal inflammation is selected from thegroup consisting of a muscle inflammation, myositis, a tendoninflammation, tendinitis, a ligament inflammation, a cartilageinflammation, a joint inflammation, a synovial inflammation, carpaltunnel syndrome and a bone inflammation.

According to still further features in the described preferredembodiments, the inflammation is selected from the group consisting ofan idiopathic inflammation and an inflammation of unknown etiology.

According to still further features in the described preferredembodiments, the IFN-γ is administered in a pharmaceutical compositionwhich includes a pharmaceutically acceptable carrier.

According to still further features in the described preferredembodiments, the pharmaceutically acceptable carrier adapts thecomposition for administration by a route selected from the intranasal,transdermal, intradermal, oral, buccal, parenteral, topical, rectal andinhalation route.

According to still further features in the described preferredembodiments, the carrier provides the IFN-γ in solution, suspension,emulsion, gel or skin pad.

According to still further features in the described preferredembodiments, the composition further includes a formulating agentselected from the group consisting of a suspending agent, a stabilizingagent and a dispersing agent.

According to another aspect of the present invention there is provided apharmaceutical composition for treating an inflammation in a subject inneed thereof, each dose-unit of the pharmaceutical compositioncomprising, as an active ingredient, IFN-γ in an amount of 1-800,000units, thereby ameliorating the inflammation.

According to further features in preferred embodiments of the inventiondescribed below, the amount is of 2-400,000 units.

According to still further features in the described preferredembodiments, the amount is of 5-200,000 units.

According to still further features in the described preferredembodiments, the amount is of 10-100,000 units.

According to still further features in the described preferredembodiments, the amount is of 25-50,000 units.

According to still further features in the described preferredembodiments, the amount is of 60-21,600 units.

According to still further features in the described preferredembodiments, the inflammation is associated with an inflammatorydisease, disorder or condition, the pharmaceutical composition ispackaged and identified for treatment of an inflammatory disease,disorder or condition.

According to still further features in the described preferredembodiments, the inflammation is associated with hypersensitivity, thepharmaceutical composition is packaged and identified for treatment ofhypersensitivity.

According to still further features in the described preferredembodiments, the inflammation is associated with autoimmune disease, thepharmaceutical composition is packaged and identified for treatment ofautoimmune disease.

According to still further features in the described preferredembodiments, the inflammation is associated with transplantation of agraft, the pharmaceutical composition is packaged and identified fortreatment of a disease associated with transplantation of a graft.

According to still further features in the described preferredembodiments, the inflammation is associated with an allergic disease,the pharmaceutical composition is packaged and identified for treatmentof an allergic disease.

According to still further features in the described preferredembodiments, the inflammation is associated with a neurodegenerativedisease, the pharmaceutical composition is packaged and identified fortreatment of a neurodegenerative disease.

According to still further features in the described preferredembodiments, the inflammation is associated with a cardiovasculardisease, the pharmaceutical composition is packaged and identified fortreatment of a cardiovascular disease.

According to still further features in the described preferredembodiments, the inflammation is associated with a gastrointestinaldisease, the pharmaceutical composition is packaged and identified fortreatment of a gastrointestinal disease.

According to still further features in the described preferredembodiments, the inflammation is associated with a tumor, thepharmaceutical composition is packaged and identified for treatment of atumor.

According to still further features in the described preferredembodiments, the inflammation is associated with septic shock, thepharmaceutical composition is packaged and identified for treatment ofseptic shock.

According to still further features in the described preferredembodiments, the inflammation is associated with anaphylactic shock, thepharmaceutical composition is packaged and identified for treatment ofanaphylactic shock.

According to still further features in the described preferredembodiments, the inflammation is associated with toxic shock syndrome,the pharmaceutical composition is packaged and identified for treatmentof toxic shock syndrome.

According to still further features in the described preferredembodiments, the inflammation is associated with cachexia, thepharmaceutical composition is packaged and identified for treatment ofcachexia.

According to still further features in the described preferredembodiments, the inflammation is associated with necrosis, thepharmaceutical composition is packaged and identified for treatment ofnecrosis.

According to still further features in the described preferredembodiments, the inflammation is associated with gangrene, thepharmaceutical composition is packaged and identified for treatment ofgangrene.

According to still further features in the described preferredembodiments, the inflammation is associated with a prosthetic implant,the pharmaceutical composition is packaged and identified for treatmentof a prosthetic implant.

According to still further features in the described preferredembodiments, the inflammation is associated with menstruation, thepharmaceutical composition is packaged and identified for treatment ofmenstruation.

According to still further features in the described preferredembodiments, the inflammation is associated with an ulcer, thepharmaceutical composition is packaged and identified for treatment ofan ulcer.

According to still further features in the described preferredembodiments, the inflammation is associated with an injury, thepharmaceutical composition is packaged and identified for treatment ofan injury.

According to still further features in the described preferredembodiments, the inflammation is a musculo-skeletal inflammation, thepharmaceutical composition is packaged and identified for treatment of amusculo-skeletal inflammation.

According to still further features in the described preferredembodiments, the inflammation is associated with an idiopathicinflammation, the pharmaceutical composition is packaged and identifiedfor treatment of an idiopathic inflammation.

According to still further features in the described preferredembodiments, the inflammation is associated with an inflammation ofunknown etiology, the pharmaceutical composition is packaged andidentified for treatment of an inflammation of unknown etiology.

According to still further features in the described preferredembodiments, the dose-unit achieves upon local or systemicadministration an IFN-γ bulk tissue concentration at a site ofinflammation of 1-8,000 units per kilogram body weight.

According to still further features in the described preferredembodiments, the IFN-γ bulk tissue concentration at a site ofinflammation is of 1.5-4,000 units per kilogram body weight.

According to still further features in the described preferredembodiments, the IFN-γ bulk tissue concentration at a site ofinflammation is of 2-2,000 units per kilogram body weight.

According to still further features in the described preferredembodiments, the IFN-γ bulk tissue concentration at a site ofinflammation is of 2.5-1,000 units per kilogram body weight.

According to still further features in the described preferredembodiments, the IFN-γ bulk tissue concentration at a site ofinflammation is of 3-500 units per kilogram body weight.

According to still further features in the described preferredembodiments, the IFN-γ bulk tissue concentration at a site ofinflammation is of 3.5-240 units per kilogram body weight.

According to still further features in the described preferredembodiments, the IFN-γ bulk tissue concentration at a site ofinflammation is of 4-240 units per kilogram body weight.

According to still further features in the described preferredembodiments, the pharmaceutical composition comprises, as an activeingredient, IFN-γ in an amount of 1-800,000 units, and apharmaceutically acceptable carrier adapted for systemic administration.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition for treating an inflammation in asubject in need thereof, the pharmaceutical composition comprising, asan active ingredient, IFN-γ in an amount of 0.0002-8 units, and apharmaceutically acceptable carrier adapted for local administration.

According to further features in preferred embodiments of the inventiondescribed below, the amount is of 0.0005-8 units.

According to still further features in the described preferredembodiments, the amount is of 0.001-8 units.

According to still further features in the described preferredembodiments, the amount is of 0.002-8 units.

According to still further features in the described preferredembodiments, the amount is of 0.004-8 units.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a method of using IFN-γ,without risk of causing severe side-effects, for treating a disease,disorder or condition associated with, and/or accompanied by,inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-d are histograms depicting cytofluorometric analysis ofcontrol and Ii^(−/−) splenocytes or pooled LN cells stained withanti-B220. Histograms show the percentage of B cells in each population.

FIGS. 1 e-h are dot-plots depicting cytofluorometric analysis ofsplenocytes and LN cells from control (FIGS. 1 e and 1 g) and Ii^(−/−)(FIGS. 1 f and 1 h) mice triple-stained with anti-B220, anti-IgM andanti-IgD antibody. The analysis demonstrates expression of IgM and IgDon B220⁺ gated cells. Boxes mark the immature IgD^(low) population.

FIGS. 1 i-j are dot-plots depicting cytofluorometric analysis of CD21surface expression in B220⁺ cells of control and Ii^(−/−) mice (FIGS. 1i and 1 j, respectively). The differences in IgD expression betweenFIGS. 1 e-h and FIGS. 1 i-j fall within the standard deviation and thusare not significant.

FIGS. 1 k-i are histograms depicting cytofluorometric analysis of bulksplenocytes of control and Ii^(−/−) mice (FIGS. 1 k and 1 l,respectively) double-stained with anti-B220 and anti-L-selectinantibody. Histograms depict expression of L-selectin on B220⁺ cells.

FIG. 2 a is a histogram depicting the percentage of wild-type mature Bcells (B con) or immature B cells from Ii^(−/−) mice (B Ii^(−/−))adhering to FN in the presence of PMA, LPS, SDF-1, or IL-2 stimulation.One representative experiment out of five is depicted.

FIG. 2 b is a histogram depicting percentage of control B cells adheringto fibronectin (FN) in the presence of PMA. (0.2 μg/ml), EDTA (5 mM) orcombined LDV and RGD peptides (800 μg/ml each).

FIG. 2 c is a histogram depicting the percentage of mature IgD⁺ B cellsfrom control mice (B mature con), immature Ii^(−/−) B cells (B Ii^(−/−))and immature IgD⁻ B cells from control mice (B imm con) adhering to FN.One representative experiment out of three is depicted.

FIGS. 2 d-g are histograms depicting flow cytofluorometric analysis ofintegrin expression on immature and mature B cells. Control and Ii^(−/−)splenocytes were double-stained with anti-B220 and either anti-VLA-4(FIG. 2 d), -VLA-5 (FIG. 2 e), -LFA-1 (FIG. 2 f) or α4β7 (FIG. 2 g).Histograms depict B220⁺-gated cells and depict an overlay of theexpression levels of the different integrins on control (light-coloredoutline) and Ii^(−/−) (dark-colored outline) B cells.

FIG. 2 h is a plot depicting resistance to shear stress by mature andimmature B cells adhering to FN in a short-term adhesion assay. Control(con) and Ii^(−/−) B cells were allowed to attach to plastic tissueculture dishes coated with FN, alone or co-immobilized with SDF-1 (sdf).Following attachment, flow was initiated and increased in 2- to 2.5-foldincrements every 10 s. The number of cells remaining bound at eachinterval was determined and is expressed as the percentage of inputcells remaining bound. One representative experiment out of three isdepicted.

FIG. 3 a is a histogram depicting the percentage of mature B cellsadhering to FN-coated plastic tissue culture dishes in the presence ofimmature IgD⁻ B cells from Ii^(−/−) mice mixed at different ratios inthe presence or absence of stimulation. Mature B cells (B con) wereincubated with or without labelled immature IgD⁻ B cells from Ii^(−/−)(B Ii^(−/−)) or control (B imm) mice in a 1:1 (unless otherwiseindicated) or 5:1 ratio in the presence or absence of stimulation.

FIG. 3 b is a histogram depicting the percentage of control B cellsadhering to FN-coated plastic tissue culture dishes in the presence ofconditioned medium (sup) collected from control (B con) or Ii^(−/−) (BIi^(−/−)) B cells in the presence or absence of stimulation. Onerepresentative experiment out of 7 is depicted.

FIG. 3 c is a histogram depicting the percentage of PMA-stimulatedcontrol B cells adhering to FN-coated plastic tissue culture dishes inthe presence of conditioned medium (sup) collected from control B cells(B con), unboiled or boiled Ii^(−/−) B (B Ii^(−/−)) or immature IgD⁻control B cells.

FIG. 4 a is a histogram depicting the percentage of PMA-stimulatedmature B cells adhering to FN-coated plastic tissue culture dishes inthe presence of various cytokines or in the presence of immature Bcell-conditioned medium.

FIG. 4 b is a plot depicting the percentage of PMA-stimulated mature Bcells adhering to FN-coated plastic tissue culture dishes in thepresence of different concentrations of recombinant IFN-γ.

FIG. 4 c is a histogram depicting the percentage of PMA-stimulatedmature B cells adhering to FN-coated plastic tissue culture dishes inthe presence of conditioned medium (sup) collected from Ii^(−/−) Bcells, with or without overnight treatment with rat anti-mouse IgG (ram)or rat anti-IFN-γ (a-IFN) antibody. Adhesion of cells stimulated withPMA only was designated as 100%.

FIG. 4 d depicts RT-PCR analysis of IFN-γ transcription in IgD⁻ B cellsof Ii^(−/−) control mice and in IgD⁺ B cells of control mice.

FIG. 4 e depicts RT-PCR analysis of IFN-γ transcription in IgD⁺, IgD⁻CD21⁺ and IgD⁻ CD21⁻B cells of control mice.

FIG. 4 f depicts RT-PCR analysis of IFN-γ transcription in IgD⁻ CD21⁻and IgD⁺ CD21⁻ B cells of Ii^(−/−) mice.

FIG. 4 g depicts IFN-γ protein expression in total cell lysates of IgD⁻B cells from Ii^(−/−) or control mice and IgD⁺ B cells from controlmice. The band representing IFN-γ is indicated.

FIG. 4 h depicts IFN-γ protein immunoprecipitated from conditionedmedium (sup) of control and Ii^(−/−) B cells. The protein speciescorresponding to IFN-γ is indicated.

FIG. 5 a is a histogram depicting the percentage of control B cellsmigrating to spleen or LN following treatment with conditioned mediumcollected from control (sup con) or Ii^(−/−) (sup-/-Ii) B cells, Bcell-conditioned medium treated with anti-IFN-γ antibody (sup-/-Iiα-IFN) or ultra-low levels of IFN-γ (1 unit/ml, IFN). The proportion ofcontrol cells homing to control spleen or LN in the absence ofinhibitory treatment was designated as 100%. Percent inhibition ofmigration was defined as indicated in Materials and Methods. The datashown represent the average of 3 experiments.

FIGS. 5 b-e are dot-plots depicting immunofluorescent flow cytometricanalysis of IgD and IgM surface expression in bulk splenocytes (FIGS. 5b-c) and bulk LN cells (FIGS. 5 d-e) of control (FIGS. 5 b and 5 d) andIFN-γ^(−/−) (FIGS. 5 c and 5 e) mice. The percentages of immature cellsper total B cell populations are indicated. The data depict onerepresentative mouse out of 4 analyzed.

FIG. 6 a is a histogram depicting the percentage of PMA- orSDF-1-stimulated T cells adhering to FN-coated plastic tissue culturedishes in the presence of conditioned medium (sup) collected fromcontrol (B con) or Ii^(−/−) (B Ii^(−/−)) B cells. One representativeexperiment out of 3 is depicted.

FIG. 6 b is a histogram depicting the percentage of PMA-stimulated Tcells adhering to FN-coated plastic tissue culture dishes in thepresence of 10 or 0.1 unit/ml IFN-γ. One representative experiment outof seven is depicted.

FIGS. 7 a-b are histograms depicting the percent inhibition of delayedtype hypersensitivity (DTH) response 24 h following ear-challenge ofBalb/c mice with 0.5% oxazolone in acetone/olive oil induced byinjection of Ii^(−/−) B cell-conditioned medium or by ultra-low levelsof IFN-γ. Prior to challenge, mice were injected every day (1×1) orevery two days (1×2), for 6 days, with 100 or 500 μl of either Ii^(−/−)B cell-conditioned medium (FIG. 7 a) or with IFN-γ at variousconcentrations (10, 1 and 0.1 units/ml, as indicated; FIG. 7 b). Onegroup of mice was treated with dexamethazone (dexa), ananti-inflammatory agent, as a positive control. Swellinginhibition=(thickness of treated ear in treated mouse)−(thickness ofuntreated ear in treated mouse)/(swelling without treatment)×100.

FIGS. 8 a-c depict inhibition of mononuclear cell infiltration into akidney allograft following treatment with ultra-low levels of IFN-γ.FIG. 8 a is a plot depicting relative infiltration of mononuclear cellsinto kidney tissue obtained from C3H (Syn) or Balb/c (Allo) mice on Days4 and 7 following transplantation thereof under the kidney capsule ofC3H hosts. The C3H hosts were subjected to three injections/wk of IFN-γ(5 units/day) for 1 wk prior to receiving the graft and every other dayfollowing transplantation, until time of sacrifice. Control animals(Allo) received injections of RPMI. Mononuclear infiltration was scoredby counting cells in at least four high-power fields (×100) of the renalgraft, as follows: 0—no infiltration, 1—low infiltration, 2—infiltrationonly to the allograft borders, 3—massive infiltration. Representativehigh-power fields depicting histological analysis of mononuclear cellinfiltration into Balb/c-derived kidney tissue 5 dayspost-transplantation into C3H mice treated with RPMI or low-level IFN-γare shown in FIGS. 8 b-c, respectively.

FIGS. 9 a-c are photomicrographs depicting IFN-γ mediated inhibition ofeosinophilic infiltration into asthmatic lung. Shown are untreatedBalb/c mice (FIG. 9 a) and Balb/c mice sensitized with aerosolized OVAalone (FIG. 9 b) or in combination with 5 units/d IFN-γ (FIG. 9 c).Eosinophilic infiltration was detected via Hand E staining offormalin-fixed lung tissue.

FIGS. 10 a-c are photographs depicting inhibition of TNBS-inducedcolitis in mice treated with low-levels of IFN-γ. Show arerepresentative colons from untreated mice (FIG. 10 a, control), micetreated with TNBS (FIG. 10 b, TNBS) and mice treated with TNBS+5 unitsIFN-γ per day for 3 days (FIG. 10 c, TNBS+IFN).

FIG. 11 a is a histogram depicting percentage of PMA (0.2 μg/ml)stimulated 70Z/3 cells adhering to FN-coated plastic tissue culturedishes following 30 min of incubation in the presence of immature Bcell-conditioned medium (sup) or ultra-low levels of IFN-γ (0.1unit/ml).

FIG. 11 b is a histogram depicting the percentage of SDF-1 (50 μg/ml)stimulated 70Z/3 cells adhering to FN-coated plastic tissue culturedishes following 30 min of incubation in the presence of differentconcentrations of IFN-γ, as indicated (units/ml). One representativeexperiment out of five is depicted

FIG. 11 c is a histogram depicting the percentage of 70Z/3 cellsmigrating towards SDF-1 in 24-well transwell plates following 30 min ofincubation in the presence of immature B cell-conditioned medium (sup)or different concentrations of IFN-γ, as indicated (units/ml). The datadepict one representative experiment out of three.

FIG. 12 a is a histogram depicting the percentage of PMA (0.2 μg/ml)stimulated 70Z/3 cells adhering to FN-coated plastic tissue culturedishes following 30 min of incubation in the presence of immature Bcell-conditioned medium (sup) or ultra-low levels of IFN-γ (0.1 unit/ml)following pre-treatment for 2.5 h with anti-CD8 or -IFNR antibody. Onerepresentative experiment out of three is depicted.

FIG. 12 b is a histogram depicting the percentage of 70Z/3 cellsmigrating towards SDF-1 in 24-well transwell plates following 30 min ofincubation in the presence or absence of ultra-low levels of IFN-γ (0.1unit/ml) following pre-treatment for 3 h with anti-CD8 or -IFNRantibody. Migration was analyzed by immunofluorescent flow cytometry andthe data depict one representative experiment out of three.

FIG. 13 a is a histogram depicting the percentage of PMA (0.2 μg/ml)stimulated 70Z/3 cells adhering to FN following 30 min of incubation inthe presence of immature B cell-conditioned medium (sup) and wortmannin(Wo) and PD. One representative experiment out of three is depicted.

FIG. 13 b is a histogram depicting the percentage of PMA (0.2 μg/ml)stimulated 70Z/3 cells adhering to FN-coated plastic tissue culturedishes following 30 min of incubation in the presence of immature Bcell-conditioned medium (sup) or ultra-low levels of IFN-γ (0.1 unit/ml)and the PI-3-K inhibitors wortmannin (Wo) and LY294002 (Ly). Onerepresentative experiment out of three is depicted.

FIG. 13 c depicts Western immunoblotting analysis of Akt proteinphosphorylation in PMA-stimulated 70Z/3 cells cultured in FN-coatedtissue culture dishes in the presence of ultra-low levels of IFN-γ.Aliquots of 10⁷ cells were pre-incubated in the presence of absence ofultra-low levels of IFN-γ (0.1 unit/ml) for 30 min, stimulated with PMAfor 5 min following which the cells were lysed and lysate proteins weresubjected to Western immunoblotting analysis using anti-p-Akt antibody.

FIGS. 14 a-b are Western immunoblotting analyses depictingphosphorylated 85 kD protein in 70Z/3 cells pre-incubated for 30 min inthe presence of ultra-low levels of IFN-γ (0.1 unit/ml) (FIG. 14 a) orimmature B cell-conditioned medium (sup) (FIG. 14 b) and plated for 5min in FN-coated plastic tissue culture dishes in the presence of PMA.Lanes represent blotted protein from 10⁷ cells probed with anti-p-tyrantibodies.

FIGS. 14 c-d depict Western immunoblotting analyses of phosphorylatedPKCA protein in PMA-stimulated 70Z/3 cells pre-incubated for 30 min inthe presence of ultra-low levels of IFN-γ (0.1 unit/ml) or immature Bcell-conditioned medium (sup) (FIG. 14 c) or in the presence ofultra-low levels of IFN-γ (0.1 unit/ml) in the presence of wortmannin(Wo) (FIG. 14 d) and plated for 5 min in FN-coated plastic tissueculture dishes in the presence of PMA. Lanes represent protein from 10⁷cells immunoprecipitated with anti-p-tyr antibody and developed withanti-PKCα antibody.

FIGS. 14 e-f depict expression of PKCα protein in purified membrane of70Z/3 cells in the presence of low-levels of IFN-γ. FIG. 14 e depictsWestern immunoblotting analysis of PKCα protein in purified membrane of70Z/3 cells incubated in the presence of IFN-γ (0.1 or 0.3 units/ml) andFIG. 14 f is a histogram depicting densitometric analysis of PKCαexpression in the Western blot shown in FIG. 14 e.

FIG. 14 g is a histogram depicting the percentage of SDF-1 or PMA (0.2μg/ml) stimulated 70Z/3 cells adhering to FN-coated plastic tissueculture dishes following pre-incubation for 1 h in the presence ofPKCα/β pseudosubstrate inhibitor. One representative experiment out ofthree is depicted.

FIG. 15 a is a histogram depicting percent actin polymerization in 70Z/3cells pre-treated with immature B cell-conditioned medium (sup) orultra-low levels of IFN-γ (0.1 unit/ml) for 30 min and subsequentlystimulated with SDF-1. Following this treatment, the cells were fixedand permeabilized, intracellular F-actin was stained withFITC-phalloidin and labelled cells were analyzed by immunofluorescentflow cytometry.

FIG. 15 b is a histogram depicting percent actin polymerization in 70Z/3cells pre-treated with PKC inhibitors for 1 h and subsequentlystimulated with SDF-1. Following this treatment, the cells were fixedand permeabilized, intracellular F-actin was stained withFITC-phalloidin and labelled cells were analyzed by immunofluorescentflow cytometry.

FIG. 15 c is a histogram depicting percent actin polymerization in 70Z/3cells pre-treated with IFN γ (0.1 unit/ml) in the presence of wortmannin(wor) for 30 min and subsequently stimulated with SDF-1. Following thistreatment, the cells were fixed and permeabilized, intracellular F-actinwas stained with FITC-phalloidin and labelled cells were analyzed byimmunofluorescent flow cytometry.

FIG. 15 d is a histogram depicting percent actin polymerization in 70Z/3cells pre-treated with IFN γ (0.1 or 1 unit/ml) in the presence ofLY29004 (LY) for 30 min and subsequently stimulated with SDF-1.Following this treatment, the cells were fixed and permeabilized,intracellular F-actin was stained with FITC-phalloidin and labelledcells were analyzed by immunofluorescent flow cytometry.

FIG. 16 a is a histogram depicting percent actin polymerization inprimary B cells pre-treated with ultra-low levels of IFN-γ (0.1 unit/ml)in the presence of LY for 30 min and subsequently stimulated with SDF-1.Following this treatment, the cells were fixed and permeabilized,intracellular F-actin was stained with FITC-phalloidin and labelledcells were analyzed by immunofluorescent flow cytometry.

FIG. 16 b is a histogram depicting percent actin polymerization inprimary B cells pre-treated for 1 h with PKCα/β or CaM kinase IIinhibitors (CaM) and subsequently stimulated with SDF-1. Following thistreatment, the cells were fixed and permeabilized, intracellular F-actinwas stained with FITC-phalloidin and labelled cells were analyzed byimmunofluorescent flow cytometry.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods and compositions which can be usedto treat an inflammation in a subject in need thereof. Specifically, thepresent invention employs ultra-low levels of IFN-γ to treat a range ofdiseases associated with, and/or accompanied by, inflammation such as,but not limited to, graft rejection, allergy, autoimmune disease andhypersensitivity.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or exemplified in the Examples. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Inflammatory processes, whether normal or pathological, require adhesionof lymphocytes to the ECM so as to enable their extravasation, migrationand infiltration. Such processes are critical for the maturation,antigenic activation and effector responses of lymphocytes which, inturn, are required to mediate pro-inflammatory functions. One approachwhich has been attempted in the prior art to treat diseases associatedwith, and/or accompanied by, inflammation has been to employ high dosesof the cytokine IFN-γ which, however was shown to lead to side-effectsof unacceptable severity.

Various methods of employing IFN-γ to treat diseases associated with,and/or accompanied by, inflammation have been described by the priorart.

For example, as described in the Background section, prior art treatmentof asthma in mice required administration of at least 800,000 unitsIFN-γ per kilogram body weight for effective suppression of disease.

Furthermore, in humans, as described in the Background section above,prior art treatment of the inflammatory diseases idiopathic pulmonaryfibrosis and Crohn's disease was attempted with doses of 33,000 and15,000 units of IFN-γ per kilogram body weight, respectively. Such highlevels of IFN-γ, however, caused side-effects of such severity as toprohibit their use in humans.

Activity units of IFN-γ are titrated by manufacturers of this cytokinevia an anti-viral activity assay wherein 1 unit/ml of interferon isdefined as the concentration necessary to produce a cytopathic effect of50% [Rubinstein, S., Familletti, P. C., and Pestka, S. (1981)“Convenient Assay for Interferons,” J. Virol. 37, 755-758; Familletti,P. C., Rubinstein, S., and Pestka, S. (1981)” A Convenient and RapidCytopathic Effect Inhibition Assay for Interferon,” in Methods inEnzymology, Vol. 78 (S. Pestka, ed.), Academic Press, New York,387-394].

Thus, all prior art approaches employing IFN-γ have failed to provideadequate solutions for treating diseases associated with, and/oraccompanied by, inflammation without risk of severe side-effects.

While conceiving the present invention it was hypothesized that suitablylow doses of IFN-γ could be employed for effectively treating diseasesassociated with, and/or accompanied by, inflammation while beingsufficiently low so as to avoid causing the prohibitively severeside-effects caused by prior art levels thereof.

While reducing the present invention to practice it was unexpectedlyuncovered, as described in detail in Example 2 of the Examples sectionbelow, that levels of IFN-γ as low as 4 units per kilogram body weight(0.1 units IFN-γ administered systemically to a 25 g mouse) could beemployed to effectively treat DTH, that levels of IFN-γ as low as 200units per kilogram body weight (5 units IFN-γ administered systemicallyto a 25 g mouse) could be employed to effectively treat delayed-typehypersensitivity, allograft rejection and colitis and that levels ofIFN-γ, as low as 240 units per kilogram body weight (6 units IFN-γadministered systemically to a 25 g mouse) could be employed to veryeffectively suppress asthma.

Hence, the method of the present invention can effectively treat a rangeof diseases associated with inflammation via levels of IFN-γ two to fourorders of magnitude lower than the lowest levels employed in the priorart and, as such, the method of the present invention represents aradical improvement over the prior art.

Thus, according to one aspect of the present invention, there isprovided a method of treating an inflammation in a subject in needthereof, which method is effected by administering to the subjectultra-low levels of IFN-γ, ultra-low levels of IFN-γ being definedherein as levels administered at 1-8000 units per kilogram body weight.

According to the present invention, IFN-γ is administered at levels suchas, but not limited to, 1-8,000, 0.2-2,000, 0.5-1,000, 1-500, 2-240 and4-240 units per kilogram body weight to treat diseases associated with,and/or accompanied by, inflammation.

According to a further aspect of the present invention, IFN-γ at suchlevels is administered in a pharmaceutical composition which ispreferably packaged and identified for treatment of an inflammatorydisease, disorder or condition.

According to a preferred embodiment of the present invention, thepharmaceutical composition includes a pharmaceutically acceptablecarrier adapting the composition for local or systemic administration bya route such as, but not limited to, the intranasal, transdermal,intradermal, oral, buccal, parenteral, topical, rectal or inhalationroutes.

Preferably, IFN-γ is administered via the parenteral route, such as, forexample, via injection, as described below in Example 2 of the Examplessection.

According to the method of the present invention, the pharmaceuticallyacceptable carrier consists of, for example, a solution, a suspension,an emulsion, a gel or a skin pad.

According to an embodiment of the present invention, the pharmaceuticalcomposition further includes a formulating agent such as, but notlimited to, a suspending agent, a stabilizing agent and a dispersingagent.

Preferably, the pharmaceutically acceptable carrier provides IFN-γ insolution.

According to a preferred embodiment of the present invention thepharmaceutically acceptable carrier is adapted for systemic or localadministration.

Preferably, IFN-γ is administered systemically, as described in Example2 of the Examples section, below.

Thus, according to a further aspect of the present invention, thedose-unit for systemic administration of the pharmaceutical compositionof the present invention comprises, as an active ingredient, IFN-γ in anamount, such as, but not limited to, 1-800,000, 2-400,000, 5-200,000,10-100,000, 25-50,000 and 60-21,600 units.

Preferably, the dose-unit for systemic administration of thepharmaceutical composition of the present invention comprises, as anactive ingredient, IFN-γ in an amount of 60-21,600 units. Such a rangecovers a minimal dose corresponding to 4 units per kilogram in a 15kilogram individual to a maximal dose corresponding to 240 units perkilogram in a 90 kilogram individual.

Alternately, the dose-unit for local administration of thepharmaceutical composition of the present invention comprises, as anactive ingredient, IFN-γ in an amount, such as, but not limited to,0.0002-8, 0.0005-8, 0.001-8, 0.002-8 and 0.004-8 units.

Preferably, the dose-unit for local administration of the pharmaceuticalcomposition of the present invention comprises, as an active ingredient,IFN-γ in an amount of 0.004-8 units. Such a range covers a minimal localdose corresponding to a concentration of 4 units per kilogram bulktissue in 1 cc of bulk tissue to a maximal local dose corresponding to aconcentration of 240 units per kilogram bulk tissue in 100 cc of bulktissue.

Thus, according still a further aspect of the present invention, thedose-unit of the pharmaceutical composition of the present inventionachieves, upon administration, an IFN-γ bulk tissue concentration at asite of inflammation, such as, but not limited to, 1-8,000, 1.5-4,000,2-2,000, 2.5-1,000, 3-500, 3.5-240 and 4-240 units per kilogram bodyweight.

Preferably, according to the method of the present invention, IFN-γ isadministered so as to achieve a concentration of 4-240 units perkilogram body weight at a site of inflammation. The concentrationsranging between 4-240 units per kilogram body weight were determined,while reducing the present invention to practice, as being optimal fortreatment of various diseases associated with, and/or accompanied by,inflammation, as described hereinabove and as described in furtherdetail in Example 2 of the Examples section below.

According to the method of the present invention, treatment of aninflammation with IFN-γ is effected by administering IFN-γ at levelssufficiently low so as to avoid the severe side-effects associated withprior art administration of high levels of IFN-γ, as describedhereinabove.

Examples of such unwanted side-effects include, but are not limited to,fever, chills, flu symptoms, bone pain, muscle pain, anorexia, fatigue,nausea, vomiting, leukopenia, diarrhea, fatigue, abnormal liverfunction, black, tarry stools; blood in urine, blood in stools,confusion, cough, hoarseness, loss of balance control, mask-like face,painful urination, difficult urination, pinpoint red spots on skin,shuffling walk, stiffness of arms, stiffness of legs, trembling ofhands, shaking of hands, trembling of fingers, shaking of fingers,trouble in speaking, trouble in swallowing, trouble in thinking, troublein concentrating, trouble in walking, unusual bleeding, unusualbruising, general feeling of discomfort, general feeling of illness,headache, skin rash, unusual tiredness, back pain, side-pain, dizziness,joint pain, loss of appetite and weight loss.

According to an embodiment of the present invention, IFN-γ is used totreat inflammation associated with inflammatory diseases, disorders orconditions.

Examples of inflammatory diseases include, but are not limited to,chronic inflammatory diseases and acute inflammatory diseases.

According to one preferred embodiment of the present invention IFN-γ isemployed to treat chronic inflammatory disease, such as colitis, asdescribed in Example 2 of the Examples section, below.

According to another preferred embodiment of the present invention,IFN-γ is employed to treat acute inflammatory disease, such as asthma,as described in Example 2 of the Examples section, below.

According to yet a further embodiment, IFN-γ is used to treat aninflammation associated with hypersensitivity.

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

According to one embodiment of the present invention, IFN-γ is employedto treat Type I or immediate hypersensitivity, such as asthma, asdescribed hereinabove and as further described in Example 2 of theExamples section, below.

Examples of Type II hypersensitivity include, but are not limited to,rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoidarthritis (Krenn V. et al., Histol Histopathol July 2000; 15 (3): 791),spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases,systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17(1-2): 49), sclerosis, systemic sclerosis (Renaudineau Y. et al., ClinDiagn Lab Immunol. March 1999; 6 (2): 156); Chan O T. et al., ImmunolRev June 1999; 169: 107), glandular diseases, glandular autoimmunediseases, pancreatic autoimmune diseases, diabetes, Type I diabetes(Zimmet P. Diabetes Res Clin Pract October 1996; 34 Suppl:S125), thyroiddiseases, autoimmune thyroid diseases, Graves' disease (Orgiazzi J.Endocrinol Metab Clin North Am June 2000; 29 (2): 339), thyroiditis,spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J ImmunolDec. 15, 2000; 165 (12): 7262), Hashimoto's thyroiditis (Toyoda N. etal., Nippon Rinsho August 1999; 57 (8): 1810), myxedema, idiopathicmyxedema (Mitsuma T. Nippon Rinsho. August 1999; 57 (8): 1759);autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity(Garza K M. et al., J Reprod Immunol February 1998; 37 (2): 87),autoimmune anti-sperm infertility (Diekman A B. et al., Am J ReprodImmunol. March 2000; 43 (3): 134), repeated fetal loss (Tincani A. etal., Lupus 1998; 7 Suppl 2: S107-9), neurodegenerative diseases,neurological diseases, neurological autoimmune diseases, multiplesclerosis (Cross A H. et al., J Neuroimmunol Jan. 1, 2001; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997;49: 77), myasthenia gravis (Infante A J. And Kraig E, Int Rev Immunol1999; 18 (1-2): 83), motor neuropathies (Kornberg A J. J Clin Neurosci.May2000; 7 (3): 191), Guillain-Barre syndrome, neuropathies andautoimmune neuropathies (Kusunoki S. Am J Med Sci. April 2000; 319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (TakamoriM. Am J Med Sci. April 2000; 319 (4): 204), paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressivecerebellar atrophies, encephalitis, Rasmussen's encephalitis,amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourettesyndrome, polyendocrinopathies, autoimmune polyendocrinopathies (AntoineJ C. and Honnorat J. Rev Neurol (Paris) January 2000; 156 (1): 23);neuropathies, dysimmune neuropathies (Nobile-Orazio E. et al.,Electroencephalogr Clin Neurophysiol Suppl 1999; 50: 419);neuromyotonia, acquired neuromyotonia, arthrogryposis multiplexcongenita (Vincent A. et al., Ann N Y Acad Sci. May 13, 1998; 841: 482),cardiovascular diseases, cardiovascular autoimmune diseases,atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2: S135),myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2: S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2: S107-9),granulomatosis, Wegener's granulomatosis, arteritis, Takayasu'sarteritis and Kawasaki syndrome (Praprotnik S. et al., Wien KlinWochenschr Aug. 25, 2000; 112 (15-16): 660); anti-factor VIII autoimmunedisease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26 (2):157); vasculitises, necrotizing small vessel vasculitises, microscopicpolyangiitis, Churg and Strauss syndrome, glomerulonephritis,pauci-immune focal necrotizing glomerulonephritis, crescenticglomerulonephritis (Noel L H. Ann Med Interne (Paris). May 2000; 151(3): 178); antiphospholipid syndrome (Flamholz R. et al., J ClinApheresis 1999; 14 (4): 171); heart failure, agonist-likebeta-adrenoceptor antibodies in heart failure (Wallukat G. et al., Am JCardiol. Jun. 17, 1999; 83 (12A): 75H), thrombocytopenic purpura (MocciaF. Ann Ital Med Int. April-June 1999; 14 (2): 114); hemolytic anemia,autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma January1998; 28 (3-4): 285), gastrointestinal diseases, autoimmune diseases ofthe gastrointestinal tract, intestinal diseases, chronic inflammatoryintestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol.January 2000; 23 (1): 16), celiac disease (Landau Y E. and Shoenfeld Y.Harefuah Jan. 16, 2000; 138 (2): 122), autoimmune diseases of themusculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist E.et al., Int Arch Allergy Immunol September 2000; 123 (1): 92); smoothmuscle autoimmune disease (Zauli D. et al., Biomed Pharmacother June1999; 53 (5-6): 234), hepatic diseases, hepatic autoimmune diseases,autoimmune hepatitis (Manns M P. J Hepatol August 2000; 33 (2): 326) andprimary biliary cirrhosis (Strassburg C P. et al., Eur J GastroenterolHepatol. June 1999; 11 (6): 595).

According to a yet another embodiment, the method of the presentinvention is employed to treat Type IV or T lymphocyte mediatedhypersensitivity.

Preferably, the method of the present invention is employed to treatType IV or T lymphocyte mediated hypersensitivity such as DTH, asdescribed hereinabove and as described in greater detail in Example 2 ofthe Examples section, below.

Examples of Type IV or T cell mediated hypersensitivity, include, butare not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R,McDevitt H O. Proc Natl Acad Sci USA Jan. 18, 1994; 91 (2): 437),systemic diseases, systemic autoimmune diseases, systemic lupuserythematosus (Datta SK., Lupus 1998; 7 (9): 591), glandular diseases,glandular autoimmune diseases, pancreatic diseases, pancreaticautoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth G S.Ann. Rev. Immunol. 8: 647); thyroid diseases, autoimmune thyroiddiseases, Graves' disease (Sakata S. et al., Mol Cell Endocrinol March1993; 92 (1): 77); ovarian diseases (Garza K M. et al., J Reprod ImmunolFebruary 1998; 37 (2): 87), prostatitis, autoimmune prostatitis(Alexander RB. et al., Urology December 1997; 50 (6): 893),polyglandular syndrome, autoimmune polyglandular syndrome, Type Iautoimmune polyglandular syndrome (Hara T. et al., Blood. Mar. 1, 1991;77 (5): 1127), neurological diseases, autoimmune neurological diseases,multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al., JNeurol Neurosurg Psychiatry May 1994; 57 (5): 544), myasthenia gravis(Oshima M. et al., Eur J Immunol December 1990; 20 (12): 2563), stiffmansyndrome (Hiemstra H S. et al., Proc Natl Acad Sci USA Mar. 27, 2001; 98(7): 3988), cardiovascular diseases, cardiac autoimmunity in Chagas'disease (Cunha-Neto E. et al., J Clin Invest Oct. 15, 1996; 98 (8):1709), autoimmune thrombocytopenic purpura (Semple J W. et al., BloodMay 15, 1996; 87 (10): 4245), anti-helper T lymphocyte autoimmunity(Caporossi A P. et al., Viral Immunol 1998; 11 (1): 9), hemolytic anemia(Sallah S. et al., Ann Hematol March 1997; 74 (3): 139), hepaticdiseases, hepatic autoimmune diseases, hepatitis, chronic activehepatitis (Franco A. et al., Clin Immunol Immunopathol March 1990; 54(3): 382), biliary cirrhosis, primary biliary cirrhosis (Jones D E. ClinSci (Colch) November 1996; 91 (5): 551), nephric diseases, nephricautoimmune diseases, nephritis, interstitial nephritis (Kelly C J. J AmSoc Nephrol August 1990; 1 (2): 140), connective tissue diseases, eardiseases, autoimmune connective tissue diseases, autoimmune ear disease(Yoo T J. et al., Cell Immunol August 1994; 157 (1): 249), disease ofthe inner ear (Gloddek B. et al., Ann N Y Acad Sci Dec. 29, 1997; 830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skindiseases, pemphigus vulgaris, bullous pemphigoid and pemphigusfoliaceus.

Examples of delayed type hypersensitivity include, but are not limitedto, contact dermatitis and drug eruption.

Examples of types of T lymphocyte mediating hypersensitivity include,but are not limited to, helper T lymphocytes and cytotoxic Tlymphocytes.

Examples of helper T lymphocyte-mediated hypersensitivity include, butare not limited to, T_(h)1 lymphocyte mediated hypersensitivity andT_(h)2 lymphocyte mediated hypersensitivity.

As described in Example 2 of the Examples section, below, administrationof as little as 200 and 240 units IFN-γ per kilogram body weight,respectively, is used, according to the method of the present invention,to very effectively treat T_(h)1 and T_(h)2 lymphocyte mediatedhypersensitivities, such as colitis and asthma.

According to a preferred embodiment of the present invention, IFN-γ isemployed to treat an inflammation associated with an autoimmune disease.

Examples of autoimmune diseases include, but are not limited to,cardiovascular diseases, rheumatoid diseases, glandular diseases,gastrointestinal diseases, cutaneous diseases, hepatic diseases,neurological diseases, muscular diseases, nephric diseases, diseasesrelated to reproduction, connective tissue diseases and systemicdiseases.

According to another preferred embodiment of the present invention,IFN-γ is employed to treat autoimmune gastrointestinal diseases, such ascolitis, as described hereinabove and as described in further detail inExample 2 of the Examples section, below.

Examples of autoimmune cardiovascular diseases include, but are notlimited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2: S132),thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2: S107-9), Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.et al., Wien Klin Wochenschr Aug. 25, 2000; 112 (15-16): 660),anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., SeminThromb Hemost. 2000; 26 (2): 157), necrotizing small vessel vasculitis,microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focalnecrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne(Paris). May 2000; 151 (3): 178), antiphospholipid syndrome (Flamholz R.et al., J Clin Apheresis 1999; 14 (4): 171), antibody-induced heartfailure (Wallukat G. et al., Am J Cardiol. Jun. 17, 1999; 83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. April-June1999; 14 (2): 114; Semple J W. et al., Blood May 15, 1996; 87 (10):4245), autoimmune hemolytic anemia (Efremov D G. et al., Leuk LymphomaJanuary 1998; 28 (3-4): 285; Sallah S. et al., Ann Hematol March 1997;74 (3): 139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. etal., J Clin Invest Oct. 15, 1996; 98 (8): 1709) and anti-helper Tlymphocyte autoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11(1): 9).

Examples of autoimmune rheumatoid diseases include, but are not limitedto rheumatoid arthritis (Krenn V. et al., Histol Histopathol July 2000;15 (3): 791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A Jan.18, 1994; 91 (2): 437) and ankylosing spondylitis (Jan Voswinkel et al.,Arthritis Res 2001; 3 (3): 189).

Examples of autoimmune glandular diseases include, but are not limitedto, pancreatic disease, Type I diabetes, thyroid disease, Graves'disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome. diseases include, but are not limited toautoimmune diseases of the pancreas, Type 1 diabetes (Castano L. andEisenbarth G S. Ann. Rev. Immunol. 8: 647; Zimmet P. Diabetes Res ClinPract October 1996; 34 Suppl: S125), autoimmune thyroid diseases,Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am June 2000;29 (2): 339; Sakata S. et al., Mol Cell Endocrinol March 1993; 92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, JImmunol Dec. 15, 2000; 165 (12): 7262), Hashimoto's thyroiditis (ToyodaN. et al., Nippon Rinsho August 1999; 57 (8): 1810), idiopathic myxedema(Mitsuma T. Nippon Rinsho. August 1999; 57 (8): 1759), ovarianautoimmunity (Garza K M. et al., J Reprod Immunol February 1998; 37 (2):87), autoimmune anti-sperm infertility (Diekman A B. et al., Am J ReprodImmunol. March 2000; 43 (3): 134), autoimmune prostatitis (Alexander RB. et al., Urology December 1997; 50 (6): 893) and Type I autoimmunepolyglandular syndrome (Hara T. et al., Blood. Mar. 1, 1991; 77 (5):1127).

Examples of autoimmune gastrointestinal diseases include, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol Hepatol. January 2000; 23 (1): 16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah Jan. 16, 2000; 138 (2): 122),colitis, ileitis and Crohn's disease.

Examples of autoimmune cutaneous diseases include, but are not limitedto, autoimmune bullous skin diseases, such as, but are not limited to,pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

Examples of autoimmune hepatic diseases include, but are not limited to,hepatitis, autoimmune chronic active hepatitis (Franco A. et al., ClinImmunol Immunopathol March 1990; 54 (3): 382), primary biliary cirrhosis(Jones D E. Clin Sci (Colch) November 1996; 91 (5): 551; Strassburg C P.et al., Eur J Gastroenterol Hepatol. Jun. 1999; 11 (6): 595) andautoimmune hepatitis (Manns M P. J Hepatol August 2000; 33 (2): 326).

Examples of autoimmune neurological diseases include, but are notlimited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol Jan.1, 2001; 112 (1-2): 1), Alzheimer's disease (Oron L. et al., J NeuralTransm Suppl. 1997; 49: 77), myasthenia gravis (Infante A J. And KraigE, Int Rev Immunol 1999; 18 (1-2): 83; Oshima M. et al., Eur J ImmunolDecember 1990; 20 (12): 2563), neuropathies, motor neuropathies (KombergA J. J Clin Neurosci. May 2000; 7 (3): 191); Guillain-Barre syndrome andautoimmune neuropathies (Kusunoki S. Am J Med Sci. April 2000; 319 (4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am JMed Sci. April 2000; 319 (4): 204); paraneoplastic neurologicaldiseases, cerebellar atrophy, paraneoplastic cerebellar atrophy andstiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units SAMar. 27, 2001; 98 (7): 3988); non-paraneoplastic stiff man syndrome,progressive cerebellar atrophies, encephalitis, Rasmussen'sencephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles dela Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C.and Honnorat J. Rev Neurol (Paris) January 2000; 156 (1): 23); dysimmuneneuropathies (Nobile-Orazio E. et al., Electroencephalogr ClinNeurophysiol Suppl 1999; 50: 419); acquired neuromyotonia,arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci.May 13, 1998; 841: 482), neuritis, optic neuritis (Soderstrom M. et al.,J Neurol Neurosurg Psychiatry May 1994; 57 (5): 544) andneurodegenerative diseases.

Examples of autoimmune muscular diseases include, but are not limitedto, myositis, autoimmune myositis and primary Sjogren's syndrome (FeistE. et al., Int Arch Allergy Immunol September 2000; 123 (1): 92) andsmooth muscle autoimmune disease (Zauli D. et al., Biomed PharmacotherJune 1999; 53 (5-6): 234).

Examples of autoimmune nephric diseases include, but are not limited to,nephritis and autoimmune interstitial nephritis (Kelly C J. J Am SocNephrol August 1990; 1 (2): 140).

Examples of autoimmune diseases related to reproduction include, but arenot limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7Suppl 2: S107-9).

Examples of autoimmune connective tissue diseases include, but are notlimited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., CellImmunol August 1994; 157 (1): 249) and autoimmune diseases of the innerear (Gloddek B. et al., Ann N Y Acad Sci Dec. 29, 1997; 830: 266).

Examples of autoimmune systemic diseases include, but are not limitedto, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2): 49) and systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. March 1999; 6 (2): 156); Chan O T. et al., Immunol Rev June1999; 169: 107).

According to an embodiment of the method of the present invention, IFN-γis employed to treat an inflammation associated with infectiousdiseases.

Examples of infectious diseases include, but are not limited to, chronicinfectious diseases, subacute infectious diseases, acute infectiousdiseases, viral diseases, bacterial diseases, protozoan diseases,parasitic diseases, fungal diseases, mycoplasma diseases and priondiseases.

According to a preferred embodiment of the method of the presentinvention, IFN-γ is employed to treat an inflammation associated with adisease associated with transplantation of a graft, as describedhereinabove and as described in further detail in Example 2 of theExamples section, below.

Examples of diseases associated with transplantation of a graft include,but are not limited to, graft rejection, chronic graft rejection,subacute graft rejection, hyperacute graft rejection, acute graftrejection and graft versus host disease.

Types of grafts whose rejection can be treated by the method of thepresent invention include, but are not limited to, syngeneic grafts,allografts and xenografts.

According to a preferred embodiment of the present invention, IFN-γ isemployed to treat allograft rejection, as described in further detail inExample 2 of the Examples section, below.

Examples of grafts include cellular grafts, tissue grafts, organ graftsand appendage grafts.

Examples of cellular grafts include, but are not limited to, stem cellgrafts, progenitor cell grafts, hematopoietic cell grafts, embryoniccell grafts and a nerve cell grafts.

Examples of tissue grafts include, but are not limited to, skin grafts,bone grafts, nerve grafts, intestine grafts, corneal grafts, cartilagegrafts, cardiac tissue grafts, cardiac valve grafts, dental grafts, hairfollicle grafts and muscle grafts.

Examples of organ grafts include, but are not limited to, kidney grafts,heart grafts, skin grafts, liver grafts, pancreatic grafts, lung graftsand intestine grafts.

Examples of appendage grafts include, but are not limited to, armgrafts, leg grafts, hand grafts, foot grafts, finger grafts, toe graftsand sexual organ grafts.

According to a preferred embodiment of the present invention, IFN-γ isemployed to treat kidney allograft rejection, as described hereinaboveand in detail in Example 2 of the Examples section, below.

According to a preferred embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammation associated withallergic diseases, as described hereinabove and as described in greaterdetail in Example 2 of the Examples section, below.

Examples of allergic diseases include, but are not limited to, asthma,hives, urticaria, pollen allergy, dust mite allergy, venom allergy,cosmetics allergy, latex allergy, chemical allergy, drug allergy, insectbite allergy, animal dander allergy, stinging plant allergy, poison ivyallergy and food allergy.

Preferably, IFN-γ is employed, according to the method of the presentinvention, to treat asthma, as described hereinabove and as described ingreater detail in Example 2 of the Examples section, below.

According to an embodiment of the method of the present invention, IFN-γis employed to treat inflammations associated with neurodegenerativediseases.

According to another embodiment of the method of the present invention,IFN-γ is employed to treat inflammations associated with cardiovasculardiseases.

According to another preferred embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammations associated withgastrointestinal diseases, as described hereinabove and as described ingreater detail in Example 2 of the Examples section, below.

Examples of gastrointestinal diseases include, but are not limited to,the examples of antibody-mediated gastrointestinal diseases listedhereinabove, the examples of T lymphocyte-mediated gastrointestinaldiseases listed hereinabove, the examples of autoimmune gastrointestinaldiseases listed hereinabove and hemorrhoids.

According to yet another preferred embodiment of the method of thepresent invention, IFN-γ is employed to treat colitis, as describedhereinabove and as described in greater detail in Example 2 of theExamples section, below.

According to still another embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammations associated withneurodegenerative diseases.

According to a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammations associated withtumors.

Examples of tumors include, but are not limited to, malignant tumors,benign tumors, solid tumors, metastatic tumors and non-solid tumors.

According to a embodiment of the method of the present invention, IFN-γis employed to treat inflammation associated with septic shock.

According to another embodiment of the method of the present invention,IFN-γ is employed to treat inflammation associated with anaphylacticshock.

According to yet another embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammation associated with toxicshock syndrome.

According to still another embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammation associated withcachexia.

According to a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammation associated withnecrosis.

According to still a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammation associated withgangrene.

According to yet a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammations associated withprosthetic implants.

Examples of prosthetic implants include, but are not limited to, breastimplants, silicone implants, dental implants, penile implants, cardiacimplants, artificial joints, bone fracture repair devices, bonereplacement implants, drug delivery implants, catheters, pacemakers,respirator tubes and stents.

According to a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammation associated withmenstruation.

According to still a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammations associated withulcers.

Examples of ulcers include, but are not limited to, skin ulcers, bedsores, gastric ulcers, peptic ulcers, buccal ulcers, nasopharyngealulcers, esophageal ulcers, duodenal ulcers, ulcerative colitis andgastrointestinal ulcers.

According to yet a further embodiment of the method of the presentinvention, IFN-γ is employed to treat inflammations associated withinjuries.

Examples of injuries include, but are not limited to, abrasions,bruises, cuts, puncture wounds, lacerations, impact wounds, concussions,contusions, thermal burns, frostbite, chemical burns, sunburns,dessications, radiation burns, radioactivity burns, smoke inhalation,torn muscles, pulled muscles, torn tendons, pulled tendons, pulledligaments, torn ligaments, hyperextensions, torn cartilage, bonefractures, pinched nerves and a gunshot wounds.

According to an embodiment of the method of the present invention, IFN-γis employed to treat musculo-skeletal inflammations.

Examples of musculo-skeletal inflammations include, but are not limitedto, muscle inflammations, myositis, tendon inflammations, tendinitis,ligament inflammations, cartilage inflammation, joint inflammations,synovial inflammations, carpal tunnel syndrome and bone inflammations.

According to an embodiment of the method of the present invention, IFN-γis employed to treat idiopathic inflammations.

According to another embodiment of the method of the present invention,IFN-γ is employed to treat inflammations of unknown etiology.

It is expected that during the life of this patent many relevant medicaldiagnostic techniques will be developed and the scope of the termanalytic mechanism is intended to include all such new technologies apriori.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8^(th) Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Ultra-Low Levels of IFN-γ Inhibit B Cell Adhesion toExtracellular Matrix and Homing to Lymph Nodes

Inflammation mediated by lymphocytes, such as B lymphocytes, asdescribed above, plays a major role in the pathogenesis of manydiseases, such as, but not limited to, autoimmune diseases, allergy,graft rejection, bacterial and viral infections and cancer. Since Blymphocytes play a central role in humoral and cellular immunity viaantibody production and antigen presentation, respectively, the abilityto downregulate B cell function is a highly desired therapeutic goal.Such downregulation may be effected, for example, by downregulation of Bcell adhesion to ECM and homing to LNs, so as to interfere withextravasation and hence activation of B cells and their migration tosites of pathogenesis. Since immature B cells are negatively selected inthe bone marrow and in the periphery before their maturation in thespleen via apoptotic elimination in response to self-antigen, naturalmechanisms exist preventing the entry of immature B cells intonon-splenic sites of foreign antigen-presentation such as LNs so as toprevent apoptosis of foreign antigen-specific clones. As demonstrated inthe experiments elaborated below, prevention of immature B cellmigration into LNs is mediated by autocrine effects of ultra-low levelsof IFN-γ, at least partly via inhibition of adhesion of activated cellsto ECM. The experiments described below in this Example furtherdemonstrate the ability of such ultra-low levels of IFN-γ to preventadhesion of B lymphocytes to ECM and migration to LNs by activatedmature B cells.

The method of the present invention therefore, harnesses thesedemonstrated anti-inflammatory properties of ultra-low levels of IFN-γto treat inflammation-related diseases, disorders or conditions whosepathogenesis involves B lymphocytes. Such therapeutic uses of ultra-lowlevels of IFN-γ represent an improvement over prior art therapeutic usesof IFN-γ since, as described above, such prior art approaches employedhigh doses of IFN-γ producing an unacceptable incidence of undesirableside-effects.

Materials and Methods:

Cells: Cells were obtained from 6-8 week-old C57BL/6 or C57BL/6J-derivedinvariant chain deficient (Ii^(−/−)) (Elliott, E. A. et al., J. Exp.Med. 1994, 179: 681) or IFN-γ deficient (IFN-γ^(−/−)) mice. Splenocytesand LN cells were purified as previously described (Elliott, E. A. etal., J. Exp. Med. 1994, 179: 681).

Purifcation of B cells: B cells were purified by treating splenocytesuspensions with anti-Thy1.2, -CD4 and -CD8 antibodies (SouthernBiotechnology Associates, USA) followed by treatment with Low Tox-Mcomplement (Cedarlane, Canada). To separate IgD⁻ from IgD⁺ cells theMACS system (Miltenyi Biotec, Auburn Calif.) was employed. The IgD⁻population was incubated with anti-CD45R (B220) antibody-conjugatedmagnetic beads and re-separated using the MACS system. For comparison ofpopulations, immature Ii^(−/−) B cells were purified by MACS and CD45Rbeads. T cells were obtained by collecting the B220⁻ population. Toseparate IgD⁻CD21⁺ from IgD⁻CD21⁻ cells, IgD⁻ cells were separatedaccording to CD21 expression using the MACS system. The IgD⁻CD21⁻ cellswere incubated with anti-CD45R magnetic beads and re-separated. Thepurity of each cell population was determined by flow cytometry.

Immunofluorescence analysis reagents: The following antibodies(Pharmingen) were employed: RA3-6B2 anti-CD45R, B3b4 anti-CD23, 7G6anti-CD21/CD35, R6-60.2 anti-IgM, AMS 9.1 anti-IgD, 7G6 anti-CD21/CD35,MEL-14 anti-L-selectin, R1-2 anti-VLA-4, 5H10-27 anti-VLA-5, 2D7anti-LFA-1 and 9C10 anti-CD49d (anti-α4).

Adhesion assays: Adhesion assays were performed as previously described(Gilat, D. et al., J Immunol. 1994, 153: 4899). Briefly, 96-well plateswere coated with FN (100 μg/ml). Cells labelled with ⁵¹Cr were suspendedin RPMI supplemented with 0.1% FCS or conditioned medium collected fromcontrol or Ii^(−/−) cells. The cells were plated at 2×10⁵ cells percoated well in the presence or absence of LPS (10 μg/ml), PMA (0.25μg/ml), IL-2 (0.2 units/μl), SDF-1 (1 μg/ml), EDTA (5 mM) or combinedLDV and RGD peptides (800 μg/ml each). After 30 min the plates werewashed, adherent cells were lysed and the released radioactivity wasdetermined. Results were expressed as the mean percentage (±SD) of boundcells from quadruplicate wells. The percentage of adherent cells wascalculated as follows: (# residual cells/well)÷(total # cellsadded/well)×100.

RNA isolation and reverse transcription: Total RNA was isolated fromcells using the Tri Reagent kit (Molecular Research Center, Cincinnati,Ohio). Reverse transcription was carried out using Superscript II RT(Gibco BRL, USA). The primers and PCR conditions employed have beenpreviously described (Reiner, S. L. et al., J. Immunol. Methods 1994,175: 275).

Detection of IFN-γ: Cells were lysed as described previously (Elliott,E. A. et al., J. Exp. Med. 1994, 179: 681). Interferon-γ wasimmunoprecipitated by overnight incubation with anti-IFN-γ monoclonalantibody (R4-6A2) (Pharmingen, USA) followed by incubation withprotein-G Sepharose beads (Pharmacia, Sweden). Crude cell lysateproteins or immunoprecipitated proteins were separated by 12% (w/v)SDS-PAGE. Separated proteins were electro-blotted onto nitrocelluloseand probed with anti-IFN-γ antibody (R4-6A2) followed by horseradishperoxidase-conjugated goat anti-rat IgG antibody (JacksonImmunoResearch).

Tracking of cells in vivo: Cells were incubated for 30 min with varioustreatments. The cells were then washed and labelled for 30 min with thefluorescent marker BCECF-AM (10 μg/ml) (Molecular Probes) with anefficiency of approximately 97%. Aliquots of 5×10⁷ washed cells wereinjected intravenously into pairs of C57BL/6 mice. After 3.5 h, spleenand LNs were removed and the percentages of fluorescent cells in bothorgans were analyzed by immunofluorescent flow cytometry. Percentinhibition of cell migration was calculated as: 100%−[(no. of cells intreated mice)÷(no. of cells in control mice)×100]. Results presented arethe average of three different experiments.

Laminar flow assays: Polystyrene plates were coated with FN (30 μg/ml;Sigma, Israel) in the presence or absence of SDF-1 (0.5 μg/ml). Theseplates were assembled to form the lower wall in a parallel wall flowchamber tube, as previously described (Lawrence, M. B. et al., Eur. J.Immunol. 1995, 25: 1025). Cells were allowed to settle in the coatedcapillary tube for 2 min before being subjected to increasing laminarflow. Cell adhesion under flow conditions was videotaped for subsequentmanual quantitation.

Cytokine concentrations: Cytokines (Gibco-BRL), were employed inadhesion assays at the following concentrations: IL-4 (10³ units/ml),IL-5 (16 units/ml), IL-6 (16 units/ml), IL-12 (3.5 ng/ml) and IFN-γ (0.1unit/ml).

Experimental Results:

Immature B cells are excluded from the LNs: To determine whetherimmature B cells are indeed excluded from secondary lymphoid organswhile recirculating through the body, profiles of B cell distribution inspleen and LNs in control and in Ii^(−/−) mice were analyzed.

In Ii^(−/−) mice there was a striking decrease in the proportion of Bcells populating the LN relative to wild-type mice (FIGS. 1 a-d andTable 1). Furthermore B cells in Ii^(−/−) mice were found to be arrestedat an immature stage, as characterized by low levels of surface IgD(FIGS. 1 e-j and Table 1) and partial CD21 surface expression (FIGS. 1i-j), as previously described (Shachar, I., and R. A. Flavell. Science1996, 274: 106; Kenty, G. et al., J. Immunol. 1998, 160: 606; Kenty, G.,and E. K. Bikoff. J. Immunol. 1999, 163: 232).

TABLE 1 Total spleen cell number and percentages of the B cellpopulation, the mature (IgD⁺) and the immature (IgD⁻) subpopulations incontrol and Ii^(−/−)mice. Cells Control Ii^(−/−) Spleen Total (9.3 ± 2)× 10⁷ (6 ± 1.6) × 10⁷ % B 46.5 ± 8.2   47 ± 10.2 % IgD⁺ 39.5 2.2 ± 1  %IgD⁻ 6.8 44.5 LN % B 36 ± 3 15 ± 4 % IgD⁺ 35 13.3 % IgD⁻ 1  1.6

These observations confirm that immature B cells are depleted from theLN, even though the Ii^(−/−) population was found to express significantsurface levels of the endothelial adhesion molecule L-selectin (FIGS. 1k-l).

Unlike mature B cells, activated B cells displaying an immaturephenotype do not adhere to ECM: To migrate to the LNs, leukocytes, suchas B cells, must interact with adhesive components of the ECM (Butcher,E. C., and L. J. Picker. Science 1996, 272: 60). Therefore the adhesionresponse of activated mature (control) and immature (Ii^(−/−)) Bsplenocytes was compared by studying their ability to adhere to FN, amajor ECM-based integrin ligand, as follows.

Cells labelled with ⁵¹Cr were plated for 30 min on FN-coated microtiterwells in the presence of the cell activator PMA, a potent agonist ofintegrin-mediated adhesion (Shimizu, Y. et al., J. Exp. Med. 1992, 175:577; Faull, R. J. et al., J. Exp. Med. 1994, 179: 1307), LPS, a B cellmitogen, or SDF-1, a potent B cell chemoattractant (Bleul, C. C. et al.,J. Exp. Med. 1998, 187 187: 753) and adhesion was assessed.

Following stimulation, control B cells were found to dramaticallyincrease their adhesion to FN, whereas in contrast, the adhesion ofIi^(−/−) B cells remained unchanged (FIG. 2 a). As expected, B cells didnot respond to stimulation with IL-2. The adhesion of B lymphocytes toFN in response to stimulation was observed to be abrogated in thepresence of the integrin-inhibitor EDTA, as well as by combined LDV andRGD peptides, selective blockers of VLA-4 and VLA-5 integrins (FIG. 2b). Thus, the observed adhesion response was integrin-mediated.

In order to determine whether the inability of Ii^(−/−) B cells toincrease adhesion to FN was due to the developmental stage of the cellsor to Ii deficiency, the adherence of purified control IgD⁻ and Ii^(−/−)B cells in response to SDF-1 or PMA stimulation was compared. Like Bcells derived from Ii^(−/−) mice, IgD⁻ B cells from control mice wereunable to increase their adhesion to FN in response to stimulation (FIG.2 c). Thus, the stage of maturity, and not the lack of Ii, determinedthe response. Similar cell surface expression levels of the integrinsVLA-4, VLA-5, LFA-1 and α4, the major FN receptors, were detected onboth Ii^(−/−) and control B cells (FIGS. 2 d, 2 e, 2 f and 2 g,respectively). Thus, immature B cells do not adhere to the ECM eventhough they possess the adhesive molecules required for such adherence.

To verify that intrinsic integrin function was not impaired, bothcontrol and Ii^(−/−) populations were tested in a short-term adhesionassay on FN-coated surfaces assembled in a flow chamber apparatus. Inthe absence of stimulation, Ii^(−/−) B cells were shown to exhibit aslightly stronger spontaneous adhesion to FN than that of the controlcells. Surprisingly, rapid adhesion stimulation to FN was induced inboth cell types by a 2 min treatment with SDF-1 (FIG. 2 h) or PMA (notshown).

These results indicated that both the expression of integrin receptorsfor FN and their ability to respond to rapid stimulation are normal inimmature B cells. Thus, the lack of adhesion by immature cells duringthe 30 min adhesion assay appeared to result from inhibitory eventssubsequent to a normal initial integrin-mediated adhesion.

B cells displaying an immature phenotype prevent ECM adhesion ofco-mixed mature B cells: To test whether the inhibition of adhesionmediated by immature cells is transferable to other cells, the abilityof purified control immature IgD⁻ or Ii^(−/−) B cells to inhibit theadhesion of purified mature IgD⁺ B cells when co-mixed was analyzed.Both the control IgD⁻ and the Ii^(−/−) immature B cells were found tohave an inhibitory effect on the adhesion of mature B-lymphocytes to FNwhen co-mixed in a 1:1 ratio (FIG. 3 a). This inhibition could beabrogated by a five-fold dilution of the immature cells within themature population.

B cells displaying an immature phenotype secrete a soluble factorpreventing adhesion of mature B cells to ECM: To determine whether Bcells displaying an immature phenotype inhibit adhesion mature B cellvia a secreted factor, the activity of conditioned medium collected fromB cells displaying an immature phenotype was analyzed for suchinhibitory activity, as follows.

Control IgD⁻ or Ii^(−/−) B cells were plated in FN-coated microwells for30 min and the conditioned medium was collected and tested for itsability to inhibit the adhesion of activated control B cells to FN.Whereas control B cell-conditioned medium was found not to affect celladhesion, the adhesion response of cells incubated with Ii^(−/−) Bcell-conditioned medium (FIGS. 3 b-c) or control IgD⁻ B cell-conditionedmedium (FIG. 3 c) was inhibited following stimulation. Thus, immature Bcell-conditioned medium was found to contain a soluble factor(s)inhibiting the adhesion of mature B cells. This factor(s) wasfurthermore determined to be heat sensitive (FIG. 3 c).

Ultra-low levels of IFN-γ prevent adhesion of B cells to FN: Todetermine the identity of the soluble factor(s), various cytokines werescreened for their ability to suppress adhesion of activated B cells toFN. Of the chemokines tested, only IFN-γ, optimally at ultra-low levels(0.1 unit/ml), was observed to mimic the effect of immature Bcell-conditioned medium (FIG. 4 a). Such effect was shown to be mediatedin a concentration dependent manner (FIG. 4 b). Neutralizing anti-IFN-γantibody was demonstrated to substantially block such an inhibitoryeffect thereby confirming the capacity of IFN-γ to inhibit adhesion ofactivated B cells to the ECM (FIG. 4 c).

IFN-γ is selectively expressed in B cells displaying an immaturephenotype: RT-PCR analysis revealed that IFN-γ expression by immature Bcells is developmentally regulated at the transcriptional level. WhereasIFN-γ mRNA was detected in both control IgD⁻ and Ii^(−/−) B cells, thistranscript was not found to transcribed in control IgD⁺ B cells (FIG. 4d). Furthermore, the low frequency, partially mature IgD⁺ B cellpopulation present in Ii^(−/−) mice was found to downregulate IFN-γ mRNAlevels as well (data not shown).

In splenic lymphoid follicles, newly immigrant immature B cells can beseparated from marginal zone cells by differential expression of theCD21 marker, thus in order to determine whether these populationsdisplay distinct IFN-γ expression profiles, transcription of IFN-γ inCD21 positive and negative IgD⁻ populations was analyzed.

As can be seen in FIGS. 4 e and 4 f, respectively, IFN-γ mRNA can bedetected in the IgD⁻ CD21⁻ cells of both control and Ii^(−/−) mice.Thus, B cells downregulate IFN-γ transcription following acquisition ofCD21, indicating that IFN-γ transcription is transcribed in immature Bcells newly arrived in the spleen.

Western immunoblotting analysis of IFN-γ protein expression in B cellsduring maturation revealed maturation-dependent regulation of IFN-γproduction and secretion. Immature (IgD⁻) B cells of control or Ii^(−/−)mice were found to express significantly higher levels of IFN-γ proteinthan IgD⁺ cells (FIG. 4 g). Importantly, Ii^(−/−) cell-conditionedmedium was found to contain significant levels of IFN-γ protein,indicating that IFN-γ is both produced and secreted by immature B cells(FIG. 4 h).

These results therefore demonstrated that IFN-γ is selectivelytranscribed, translated and secreted by B cells displaying an immaturephenotype.

Ultra-low levels of IFN-γ inhibit migration of B cells to LNs: Theeffects of immature B cell-conditioned medium or ultra-low levels ofIFN-γ on migration of normal B cells into LNs in vivo were investigated.

Bulk splenocytes of control or Ii^(−/−) mice were treated withnon-conditioned, control B cell-conditioned, Ii^(−/−) B cell-conditionedmedium, Ii^(−/−) B cell-conditioned medium treated with anti-IFN-γantibody or ultra-low levels of IFN-γ (1 unit/ml). Treated cells werewashed, labelled with the fluorescent dye BCECF-AM and equal numbers oflive cells were injected intravenously into control mice. The proportionof labelled cells recovered in the spleen and LNs was determined 3.5 hfollowing injection.

The results obtained are depicted in FIG. 5 a. The extent of labelledcell accumulation in the spleen was found to be unaffected by thevarious pre-treatments. In contrast, migration of Ii^(−/−) and ofcontrol cells treated with Ii^(−/−) B cell-conditioned medium orultra-low levels of IFN-γ (1 unit/ml) to the LN was observed to besignificantly decreased, whereas cells treated with control Bcell-conditioned medium or non-conditioned medium, exhibited a degree ofmigration similar to untreated cells. Furthermore, when control cellswere incubated in Ii^(−/−) B cell-conditioned medium in the presence ofneutralizing anti-IFN-γ antibody, the ability of this medium to inhibithoming of the cells to the LNs was substantially inhibited, thusconfirming that the inhibitory effect of immature B cell-conditionedmedium on homing of B cells to LNs is mediated by IFN-γ.

In order to confirm the biological significance of these results, thematuration profiles of B cells in spleen and LN of IFN-γ^(−/−) mice wereanalyzed. While spleens of both types of mice were found to containsimilar proportions of immature and mature B cells (FIGS. 5 b and 5 c,respectively), LNs of IFN-γ^(−/−) mice were found to be populated with asignificantly greater proportion of immature B cells than control Bcells (FIGS. 5 e and 5 d, respectively). These results thereforeindicated a physiological role for IFN-γ in preventing retention ofimmature B cells in secondary lymphoid organs, such as LNs.

In summary, these experiments demonstrated the ability of Ii^(−/−) Bcell-conditioned medium or ultra-low levels of IFN-γ to inhibit ECMadhesion and LN migration of B cells. Such capacity of ultra-low levelsof IFN-γ to inhibit B cell functionality therefore demonstrates theeffectiveness of the method of the present invention to treatinflammation-related disorders whose pathogenesis involves Blymphocytes. Therapeutic use of ultra-low levels of IFN-γ, according tothe method of the present invention, represents a marked improvementover prior art therapeutic use of IFN-γ, employing high levels of thiscytokine having been shown to produce an unacceptable incidence ofside-effects.

Example 2 Ultra-Low Levels of IFN-γ Inhibit ECM Adhesion and Migrationof T Lymphocytes and Constitute an Effective Treatment for TCell-Mediated Diseases

Immunoreactivity mediated by T lymphocytes, as described above, plays amajor role in the pathogenesis of many diseases, such as autoimmunediseases, allergy, graft rejection, infection and cancer. This is aconsequence of the fact that T lymphocytes are the major effectors ofspecific cellular immunity, both in their ability to respond to targetcells expressing MHC-peptide complexes and in their ability to providehelper cytokines promoting B and T lymphocyte function. Thus, theability to inhibit T lymphocyte function is a highly desired therapeuticgoal.

As demonstrated in Example 1 with respect to B lymphocytes, inhibitionof T lymphocyte effector functions can also be efficiently achieved bydownregulating their capacity to adhere to ECM and to migrate. Suchinhibition represents an effective means of preventing T lymphocytemigration to secondary lymphoid organs and inflamed tissues therebyrespectively preventing activation of T lymphocytes by professionalantigen-presenting cells and the triggering of effector function bytarget cells expressing foreign antigens in sites of inflammation.

Thus, experiments were performed, as described below, testing, andclearly demonstrating, the ability of ultra-low levels of IFN-γ toprevent ECM adhesion and migration to secondary lymphoid organ tissue byT lymphocytes, as well. Most importantly, experiments were performeddemonstrating that inhibition of T lymphocyte functions by ultra-lowlevels of IFN-γ, according to the method of the present invention, canbe very effectively employed to treat a broad range of major classes ofdiseases whose pathogenesis involves T cell-mediated immunoreactivity.

Materials and Methods:

Animals: C57BL/6 or Balb/c mice were used in experiments at 6-8 weeks ofage. All animal procedures were approved by the Animal ResearchCommittee of the Weizmann Institute of Science (Rehovot, Israel).

Isolation of organs and cells: Spleens and cells were obtained aspreviously described (Shachar, I. et al., Immunity 1995, 3: 373).Enrichment of T cells was performed using the MACS system (MiltenyiBiotec, Auburn Calif.). Spleen cells were incubated with anti-CD45RA(B220) magnetic beads and the CD45⁻ cells were collected. Helper (CD4⁺)T cells were enriched using anti-CD4 magnetic beads.

Adhesion assay: Adhesion assays were performed as previously described(Flaishon, L. et al., J. Exp. Med 2000, 192: 1381).

DTH assays: Balb/c mice were sensitized on shaved abdomens with 2%oxazolone in acetone/olive oil. Mice were injected with different dosesof immature B cell-conditioned medium or ultra-low levels of IFN-γ, oncea day or every other day of the experimental period. Delayed-typehypersensitivity was elicited 6 days later by ear-challenge with 0.5%oxazolone in acetone/olive oil and ear-thickness was measuredimmediately prior to and 24 h following challenge.

OVA sensitization and challenge: Balb/c mice were immunizedintraperitoneally on Days 0, 7, and 14 with 100 μg chicken egg OVA(Sigma, St. Louis, Mo.) mixed with 2 mg of aluminum hydroxide (Pierce,Rockford, Ill.) in 300 μl of 0.9% NaCl. Starting on Day 15 followinginitial sensitization, animals were challenged daily for 5 days (Days15-19) with 20 min inhalations of 5% OVA in 0.9% NaCl, using anultrasonic nebulizer (DeVilbiss, Somerset, Pa.) connected to a 0.5 literplastic chamber in which the animals were placed for inhalations.

The IFN-γ-treated group was injected intraperitoneally with ultra-lowlevels of murine IFN-γ (6 units in 500 μl 0.9% NaCl; Genentech, Inc.South San Francisco, Calif.) starting on Day 15 for 5 days, 5 minutesprior to each inhalation.

Measurement of AHR: AHR was measured, as previously described(Djukanovic, R. et al., Am. Rev. Respir. Dis. 1990, 142: 434), viaenhanced expiratory pause (Penn) in plethysmographic box (BuxcoElectronics, Sharon, Conn.) pressure during expiration. Briefly,conscious, spontaneously breathing animals were evaluated in closedplethysmographic chambers and pressure differences were measured betweenthe main chamber of the plethysmograph, containing the animal, and areference chamber (generating a box pressure signal). Data was expressedusing the dimensionless parameter Penn according to the formula:Penn=(Te−Tr) (PEPb)/(Tr*PIPb), where Te is the expiratory time (inseconds), Tr is the relaxation time (time of the decay of the expiratorybox pressure to 36% of peak expiratory box pressure in seconds), PEPb isthe peak expiratory pressure (in ml/s) and PIPb is the peak inspiratorypressure (in ml/s). Every animal was evaluated following the fifth OVAinhalation (early reaction) and 24 hours later (late reaction).

Bronchoalveolar lavage (BAL): BAL was performed 20 days following thelast AHR evaluation using animals deeply anesthetized with Thiopentalsod, a 22 gauge needle was inserted into the proximal trachea andsecured with a 3-0 silk suture through a midline celiotomy and animalswere euthanized by exsanguination through withdrawal of blood from theinferior vena cava. The lungs were lavaged 5 times via the trachealneedle with 1 ml of 0.9% NaCl, BAL fluid samples were collected fromeach animal and pooled and cell counts were determined using ahemocytometer. Samples were centrifuged, and pellets were resuspended in0.9% NaCl for application to Cytospin slides.

Lung histology: Lungs were prepared for histology by perfusing animalswith 20 ml of PBS followed by inflation with 1 ml of 4% formalin, untildistension. Samples were fixed in 4% formalin for 48 hr, tissues wereembedded in paraffin and 2-3 μm sections were cut and stained withhematoxylin and eosin for histochemical analysis.

Cytoskeleton rearrangement assays: T cells were pre-incubated in thepresence or absence of ultra-low levels of IFN-γ, washed, stimulated for20 s with low levels of SDF-1 (500 ng/ml), promptly fixed by incubationin three volumes of 3.7% paraformaldehyde for 10 min at roomtemperature. Fixed cells were washed and the membranes permeabilized byincubation for 2 min on ice in a solution containing HEPES (20 mM),sucrose (300 mM), NaCl (50 mM), MgCl₂ (3 mM) and Triton-X-100 (0.1%).Membrane-permeabilized cells were stained with the F-actin specificreagent FITC-phalloidin (2 μg/ml), washed with PBS, and analyzed by flowcytometry.

Hapten induced colitis: To induce colitis, anesthetized Balb/c mice weregiven enemas with a solution of trinitrobenzene sulfonic acid (TNBS)dissolved in a mixture of PBS (pH 7.2) and then mixed with an equalvolume of ethanol, yielding a solution with a final concentration of2.5% TNBS in 50% ethanol. Mice were administered a dose of 150 mgTNBS/kg body weight on Day 0 and disease severity was scored as afunction of weight loss, fur ruffling, rectal prolapse and death.

Experimental Results:

Immature B cell-conditioned medium or ultra-low levels of IFN-γabrogates activation-induced adhesion of T cells to ECM: In order tomigrate to secondary lymphoid organs, such as LNs, or to sites ofinflammation, T cells must interact extensively with the ECM (Butcher,E. C., and Picker, L. J. Science 1996, 272: 60). The method of thepresent invention therefore employs immature B cell-conditioned mediumand ultra-low levels of IFN-γ to interfere with such processes to treatdisease states. Thus, in order to demonstrate the effectiveness of sucha method, the capacity of immature B cell-conditioned medium orultra-low levels of IFN-γ to negatively regulate adhesion of T cells tothe ECM was analyzed, as described below.

Naïve T cells labelled with ⁵¹Cr were activated with PMA, a potentagonist of integrin mediated adhesion (Shimizu, Y. et al., J. Exp. Med.1992, 175: 577; Faull, R. J. et al., J. Exp. Med. 1994, 179: 1307), orSDF-1, a potent T cell chemoattractant (Bleul, C. C. et al., J. Exp.Med. 1996, 184: 1101; Bleul, C. C. et al., Nature 1996, 382: 829), inthe presence of immature B cell-conditioned medium or ultra-low levelsof IFN-γ (0.1 unit/ml). Upon activation, untreated T cells were observedto dramatically increase their adhesion to FN, whereas such adhesion bycells treated with immature B cell-conditioned medium or ultra-lowlevels of IFN-γ (0.1 unit/ml) was completely abrogated (FIGS. 6 a and 6b, respectively).

These results therefore clearly demonstrated the capacity of immature Bcell-conditioned medium or ultra-low levels of IFN-γ (0.1 unit/ml) topowerfully inhibit adhesion of activated T cells to ECM.

Treatment with immature B cell-conditioned medium or ultra-low levels ofIFN-γ inhibit migration of T cells to sites of DTH-induced inflammation:The capacity of immature B cell-conditioned medium or ultra-low levelsof IFN-γ to inhibit T cell migration to in vivo sites of inflammation,according to the method of the present invention, was analyzed byanalyzing their effect on the migration to sites of inflammation inducedby DTH, an antigen-induced cutaneous inflammatory response involvingCD4⁺ T cell-mediated pathogenesis. Delayed-type hypersensitivityexperiments were performed as follows.

Normal mice were injected with various doses of medium conditioned byincubating Ii^(−/−) B cells or ultra-low levels of IFN-γ in FN-coatedplates. Treatment of mice with Ii^(−/−) B cell-conditioned medium (FIG.7 a) or ultra-low levels of IFN-γ [FIG. 7 b; ≧4 U/kg body weight (100μl×1 unit/ml÷25 g mouse=4 U/kg)] were found to significantly inhibit earswelling, whereas control medium collected from FN-coated plates had noeffect.

These results therefore demonstrated that immature B cell-conditionedmedium or ultra-low levels of IFN-γ can be employed according to themethod of the present invention to significantly inhibit migration of Tcells to sites of inflammation in vivo and thereby ameliorate DTHresponses.

Treatment with ultra-low levels of IFN-γ significantly amelioratesallograft rejection: Transplantation of allogeneic cells, tissues andorgans is a method of treatment employed to treat a very broad range ofdisorders including many life-threatening disorders. Graft rejection,however remains a major cause of transplantation failure, regardless ofthe use of prophylactic treatments which, even when successfullyemployed, rely on powerful immunosuppressant drugs producing undesirableside-effects.

Thus, experiments to demonstrate the capacity of ultra-low levels ofIFN-γ, employed according to the method of the present invention, toameliorate allograft rejection were performed by analyzing mononuclearinfiltration into allograft tissue in treated allograft recipientanimals, as follows.

Kidney tissue from Balb/c mice (H-2d) was transplanted under the kidneycapsule of allogeneic C3H hosts (H-2k) and analyzed by histology formononuclear infiltration and tissue damage on Days 4 and 7post-transplant. The C3H hosts were treated with 3 injections oflow-dose IFN-γ (5 units/d=200 units/kg body weight/day) during the weekprior to receiving the allograft, and every other day followingtransplantation until time of sacrifice. Control animals receivedinjections of RPMI. Mononuclear infiltration was scored by countingcells in a minimum of four high-power (×100) fields in renal graftsections.

Animals treated with ultra-low levels of IFN-γ were found to displaygreatly reduced levels of mononuclear cell infiltration into allograftson Days 4 and 7 post-transplant, as quantified in FIG. 8 a. Histologicalanalysis of allograft tissue on Day 6 post-transplant in animals treatedwith ultra-low levels of IFN-γ very clearly depict a dramatic inhibitionof infiltration compared to untreated recipients (FIGS. 8 c and 8 b,respectively).

These results therefore clearly demonstrated that ultra-low levels ofIFN-γ can be effectively employed according to the method of the presentinvention to significantly ameliorate allograft rejection.

Treatment with ultra-low levels of IFN-γ alleviates asthma: Asthma, awidespread disease with often fatal consequences and whose incidence isdramatically on the increase in urban populations around the world, is achronic inflammatory disorder of the airways that is characterized byintermittent episodes of airway obstruction and wheezing, by variableairflow obstruction, airway hyperresponsiveness (AHR) and airwayinflammation. Inflammation in asthmatic lung is characterized byinfiltration of the airway wall with mast cells, lymphocytes andeosinophils. Although asthma is multifactorial in origin, recentadvances suggest that asthma is an immune disease with a prominent rolefor T lymphocytes in its pathogenesis. In particular CD4⁺ T cellsproducing a Th2 pattern of cytokines have been shown to play a pivotalrole in the pathogenesis of this disease (Huang, S. K. et al., J.Immunol. 1995, 155: 2688; Robinson, D. S. et al., N. Engl. J. Med. 1992,326: 298; Walker, C. et al., Am. Rev. Respir. Dis. 1992, 146: 109; Cohn,L. et al., J. Exp. Med. 1999, 190: 1309).

Thus, in order to demonstrate the capacity of ultra-low levels of IFN-γ,employed according to the method of the present invention, to ameliorateTh2-mediated diseases, such as asthma, experiments were performed aimedat inhibiting the migration of Th2 cells in a murine OVA-induced asthmamodel.

Inhalation of aerosolized OVA was observed to cause peribronchial andperivascular accumulation of inflammatory cells, such as eosinophils andT cells, into the tracheal submucosa of OVA-sensitized Balb/c mice,compared to non-sensitized mice (FIGS. 9 b and 9 a, respectively).However, treatment with ultra-low levels of IFN-γ (6 units/day=240units/kg body weight/day) were shown to strikingly prevent suchrecruitment of antigen-induced inflammatory cells (FIG. 9 c). Inaccordance with these observations, treatment with ultra-low levels ofIFN-γ were observed to dramatically inhibit the symptoms of asthma, suchas airflow obstruction, AHR and airway inflammation.

These results therefore showed that ultra-low levels of IFN-γ can besuccessfully employed according to the method of the present inventionto treat Th2 cell-promoted diseases, such as asthma.

Treatment with ultra-low levels of IFN-γ significantly amelioratescolitis: Autoimmune diseases of the gastrointestinal tract, such ascolitis, are debilitating diseases of widespread incidence whosetreatment using prior art techniques remains inefficient, relying on theuse of anti-inflammatory drugs, such as steroids, causing significantlevels of undesirable side-effects.

Thus, experiments were performed in order to demonstrate the capacity ofultra-low levels of IFN-γ employed according to the method of thepresent invention to treat Thl cell-mediated diseases, such as colitis,using a murine TNBS-induced colitis model in which Balb/c mice treatedwith TNBS develop potentially lethal weight loss and colitis.

As depicted by visual examination of colons, treatment of TNBS-treatedmice with ultra-low levels of IFN-γ (5 units/d=200 units/kg bodyweight/day) injected i.p. dramatically inhibited the development ofcolitis, as compared to TNBS-treated mice (FIGS. 10 c and 10 b,respectively).

These results therefore demonstrated that treatment with ultra-lowlevels of IFN-γ, according to the method of the present invention, canbe employed to effectively treat T_(h)1 lymphocyte-induced inflammatorydiseases, such as colitis.

Conclusion:

Overall the data presented in these studies demonstrated that ultra-lowdoses of IFN-γ, ranging from 4-240 units/kg body weight, have thecapacity to downregulate ECM adhesion and migration of T cells and, mostimportantly, these studies showed that the use of ultra-low levels ofIFN-γ, according to the method of the present invention, can be veryeffectively employed to treat a broad range of inflammatory disorderspathogenesis, such as allograft rejection, allergic diseases, such asasthma, caused by T_(h)2 lymphocytes, DTH and T_(h)1 lymphocyte-mediatedautoimmune diseases, such as colitis.

Thus, the method of the present invention represents a very markedimprovement over prior art uses of IFN-γ to treat diseases associatedwith, and/or accompanied by, inflammation since prior art approachesused levels of IFN-γ two to four orders of magnitude higher than thosesuccessfully employed in the present invention.

Example 3 Ultra-Low Levels of IFN-γ Downregulate Integrin-DependentAdhesion of B Cells by Activating a Pathway that Interferes withCytoskeleton Rearrangement

In the above Examples it was shown that immature B cells can activelyexclude themselves from antigen-enriched sites by downregulating theirintegrin-mediated adhesion by autocrine production of IFN-γ. Treatmentwith immature B cell-conditioned medium or ultra-low levels of IFN-γ,according to the method of the present invention, was shown to becapable of inhibiting ECM adhesion and tissue migration of both B and Tlymphocytes and thereby to effectively treat a broad range of majorclasses of inflammatory diseases, as described in Example 2.

Thus, experiments were performed in order to clearly demonstrate thebiochemical and physiological mechanisms by which ultra-low levels ofIFN-γ, employed according to the method of the present invention,inhibit the adhesion and migration of lymphocytes and thereby can beeffectively employed to treat inflammatory diseases.

Materials and Methods:

Cells: The murine pre-B cell-like lymphoma line, 70Z/3 (Paige, C.J. etal., J. Immunol. 1978, 121: 641) was grown in suspension culture at 37°C. in RPMI-1640 medium containing 10% (v/v) FCS and 200 μMβ-mercaptoethanol. Primary B cells were obtained from C57BL/6 spleensand were purified by treating splenocyte suspensions with anti-Thy1.2,-CD4, and -CD8 antibody (Southern Biotechnology Associates, USA)followed by low Tox-M complement (Cedarlane, Canada).

Immature B cell-conditioned medium: Conditioned medium was collectedfrom immature B cells from invariant chain-deficient mice (Shachar, I.,and Flavell, R. A. Science 1996, 274: 106) as previously described(Flaishon, L. et al., J. Exp. Med. 2000, 192: 1381).

Adhesion assays: Adhesion assays were performed as previously described(Flaishon, L. et al., J. Exp. Med. 2000, 192: 1381).

Transwell migration assays: Chemotaxis was assayed by using transwellchambers (6.5 mm diameter; 5 μm pores) (Corning Inc., Corning N.Y.).Approximately 5×10⁶ 70Z/3 cells were suspended in 1 ml of RPMIsupplemented with 0.25% FCS and 100 μl aliquots were placed in the uppercompartment of the transwell apparatus and the bottom chamber was filledwith 600 μl medium supplemented with 1 μg/ml SDF-1 (PeproTech, Inc.,Rocky Hill N.J.). Migration towards SDF-1 in the lower chamber wasanalyzed after 3 hours by immunofluorescent flow cytometry in thepresence or absence of invariant chain-deficient B cell-conditionedmedium or of ultra-low levels of IFN-γ. Cells were pre-incubated withanti-INF-γ receptor or anti-CD8 antibodies (Pharmingen) for 2.5 hoursprior to transwell assays in blocking experiments.

Preparation of cellular lysates: 70Z/3 cells were pre-incubated with orwithout immature B cell-conditioned medium or ultra-low levels of IFN-γin the presence of inhibitors, for 30 minutes, stimulated for 5 minuteswith PMA (0.2 μg/ml) and immediately frozen. Frozen cells were thawedand lysed in lysis buffer containing 25 mM Tris (pH 7.4), 2 mM vanadate,75 mM β-glycophosphate (pH 7.2), 2 mM EDTA, 2 mM EGTA, 10 mM NaPPi, 0.5%NP-40 and the following protease inhibitors: 10 μg/ml Leupeptin, 10μg/ml aprotinin, 10 μg/ml pepstatin, 10 μg/ml chymostatin (Roche,Switzerland), 1 mM PMSF (Sigma, Israel), and 20 mM N-ethyl-maleimide(Sigma, Israel).

Western immunoblotting analysis: Crude lysates or immunoprecipitates ofp-tyr proteins were separated by 10% (w/v) SDS-PAGE and separatedproteins were electro-blotted onto nitrocellulose membranes. Blottedproteins were probed with anti-p-AKT (Cell Signaling Technology),anti-p-tyr (pTyr99) or anti-PKCa (C-20) antibodies (Santa Cruz) andprobed blots were developed by horseradish peroxidase-conjugatedanti-mouse or anti-rabbit IgG antibodies (Jackson Labs).

Membrane purification: Cell suspension aliquots of 10⁷ cells wereincubated in PBS containing 5 μg/ml digitonin (Sigma, Israel) for 5 minon ice, centrifuged and pellets were resuspended in lysis buffer, asdescribed above.

Analysis of cytoskeletal rearrangement: Primary B cells or 70Z/3 cellswere pre-incubated for 30 min in with or without immature Bcell-conditioned medium or ultra-low levels of IFN-γ (0.1 unit/ml) inthe presence of absence of the PI-3-K inhibitors wortmannin or LY.Pre-incubated cells were washed, stimulated SDF-1 (500 ng/ml) for 20 secand immediately fixed by incubation in three volumes of 3.7%paraformaldehyde for 10 min at room temperature. Fixed cells were washedand the membranes were permeabilized for 2 min on ice in a solutioncontaining 20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl₂, and 0.1%Triton X-100. Finally, membrane-permeabilized cells were stained withthe F-actin specific reagent FITC-phalloidin (2 μg/ml), washed with PBSand analyzed by flow cytometry. The percent increase of polymerizedactin=[(mean of polymerized actin in activated cells)−(mean ofpolymerized actin in non-activated cells)]÷(mean of polymerized actin inactivated cells)×100%.

Inhibitors: The PI-3-K inhibitors (CalBiochem) and correspondingconcentrations employed were wortmannin (100 nM) and LY (200 mM). ThePKC inhibitors used were: PKCα/β pseudosubstrate (10 μM; BioMol researchlaboratories) myristoylated-AIP, CaM kinase II inhibitor (10 μg/ml;BioMol research laboratories); the PKC8 inhibitor rottlerin (5 μM;CalBiochem); and the PKC inhibitor GF109203X (0.5 μM; CalBiochem). Thep38 inhibitor SB203580 (SB) and the MEK inhibitor PD98059 (PD) were usedat concentrations of 10 μM and 25 μM, respectively.

Experimental Results:

A cell line model for inhibition of B cell migration by ultra-low levelsof IFN-γ: In order to conveniently analyze the mechanisms by whichultra-low levels of IFN-γ inhibit adhesion and migration of lymphocytesaccording to the method of the present invention, experiments wereperformed to identify a B cell line that could increase its adhesion andmigration in response to chemokine stimulation and in which suchresponses could be inhibited by ultra-low levels of IFN-γ, therebyconstituting a realistic model of such lymphocyte functions.

The 70Z/3 murine pre-B lymphoma cell line was identified as meetingthese requirements. Following PMA or SDF-1 (FIG. 10 b) stimulation,these cells were found to increase their adhesion to FN, whereasimmature B cell-conditioned medium and ultra-low levels of IFN-γ (0.1unit/ml) were found to significantly inhibit this adhesion response(FIG. 11 a). Similarly, ultra-low levels of IFN-γ (1 unit/ml) were foundto efficiently and optimally downregulate SDF-1 stimulated adhesionresponses (FIG. 11 b).

The ability of immature B cell-conditioned medium and ultra-low levelsof IFN-γ to inhibit the transwell migration of 70Z/3 cells toward thechemokine SDF-1 was also confirmed. As shown in FIG. 11 c, cellspre-incubated with immature B cell-conditioned medium and, inparticular, with ultra-low levels of IFN-γ (0.1 unit/ml) markedlydownregulated their migration toward SDF-1.

Thus, similarly to primary B cells, 70Z/3 cells respond toactivation-induced pro-adhesive stimuli and such responses in thesecells are sensitive to inhibition by immature B cell-conditioned mediumand ultra-low levels of IFN-γ. Such cells therefore constitute anappropriate model system to investigate the inhibitory signalingpathways induced by ultra-low levels of IFN-γ.

Ultra-low levels of IFN-γ inhibit lymphocyte adhesion and migration viaan IFNR-activated signaling cascade: To determine whether theIFN-γ-induced inhibition of cell adhesion and migration is initiated bythe IFN-γ receptor (IFNR) (Stark, G. R. et al., Annu. Rev. Biochem.1998, 67: 227; Bach; E. et al., Annu. Rev. Immunol. 1997, 15: 563) or bya novel receptor not previously described, the involvement of thisreceptor in the inhibitory pathway was investigated as follows.

Analysis of adhesion to FN was performed by pre-treating 70Z/3 cells for3 h with anti-IFNR or anti-CD8 antibodies, washing the cells followed bystimulation with PMA in the presence or absence of immature Bcell-conditioned medium or ultra-low levels of IFN-γ (0.1 unit/ml).

As shown in FIG. 12 a, whereas anti-CD8 control antibody did notinfluence inhibition of adhesion to FN mediated by immature Bcell-conditioned medium or by ultra-low levels of IFN-γ, neutralizinganti-IFNR antibody was observed to strikingly abrogate these inhibitoryeffects.

To further demonstrate that ultra-low levels of IFN-γ similarly inhibitcellular migration via IFNR, the transwell migration of 70Z/3 cellstowards SDF-1 was monitored in the presence of neutralizing anti-IFNRantibody.

Indeed, inhibition of cell migration towards SDF-1 mediated by ultra-lowlevels of IFN-γ (0.1 unit/ml) was found to be abrogated in cellspre-treated with neutralizing anti-IFNR antibody but not in cellspre-treated with control anti-CD8 antibody (FIG. 12 b).

These experiments therefore demonstrated that ultra-low levels of IFN-γor immature B cell-conditioned medium, employed according to the methodof the present invention, inhibit adhesion of lymphocytes via signalingcascades initiated by IFNR and, furthermore, that ultra-low levels ofIFN-γ also inhibit migration of lymphocytes via signaling cascadesinitiated by the IFNR.

Ultra-low levels of IFN-γ activate PI-3-K dependent pathways downstreamof IFNR: To monitor downstream molecules involved in inhibition oflymphocyte adhesion induced by immature B cell-conditioned medium andultra-low levels of IFN-γ, the effects of various signal transductioninhibitors on adhesion of activated lymphocytes were analyzed.

Whereas the p38 inhibitor SB (data not shown) and the MEK inhibitor PDwere not observed to affect inhibition of adhesion of PMA stimulated Blymphocytes to FN by immature B cell-conditioned medium, in the presenceof the PI-3-K inhibitor wortmannin, such inhibition of adhesion wasfully reversed (FIG. 13 a). The ability of the PI-3-K inhibitorswortmannin and LY to reverse inhibition of lymphocyte adhesion to FN byultra-low levels of IFN-γ was analyzed. As previously described, PI-3-Kinhibitors on their own were found to slightly suppress SDF-1 stimulatedadhesion of 70Z/3 cells (Wang, J. F. et al., Blood 2000, 95: 2505).However, wortmannin and LY were observed to reverse the inhibitoryeffects of immature B cell-conditioned medium and ultra-low levels ofIFN-γ (0.1 unit/ml) on adhesion of PMA-activated B lymphocytes to FN.Cells activated with PMA and treated with either immature Bcell-conditioned medium or ultra-low levels of IFN-γ (0.1 unit/ml) inthe presence of these PI-3-K inhibitors were observed to adhere to FNsimilarly to cells activated with PMA in the presence of wortmannin orLY (FIG. 13 b). These results therefore demonstrate a potential role forPI-3-K mediated signaling in such cellular adhesion.

To confirm the involvement of PI-3-K in inhibition of adhesion byultra-low levels of IFN-γ, experiments were performed in order todetermine whether the kinase Akt, a known downstream intermediate of thePI-3-K pathway (Burgering, B. M., and Coffer, P. J. Nature 1995, 376:599), is activated by such ultra-low levels of IFN-γ.

As shown in FIG. 13 c, in the presence of ultra-low levels of IFN-γ (0.1unit/ml) increased Akt phosphorylation was observed, thus providingsupport for a role for PI-3-K in mediating inhibition of lymphocyteadhesion via triggering of IFNR by ultra-low levels of IFN-γ employedaccording to the method of the present invention.

In order to identify downstream intermediates involved in signalingtriggered by ultra-low levels of IFN-γ, experiments were performed toidentify proteins whose tyrosine phosphorylation levels could be alteredin response to PMA stimulation of 70Z/3 cells and in which suchalterations could be inhibited by ultra-low levels of IFN-γ. To identifysuch candidate proteins, 70Z/3 cells were pre-incubated in the presenceor absence of immature B cell-conditioned supernatant or ultra-lowlevels of IFN-γ for 30 min, subjected to 5 min of PMA stimulation andpromptly frozen. Cells were thawed, protein lysates were prepared andthese were analyzed for the presence of tyrosine phosphorylated proteinsby Western immunoblot analysis.

An 85 kD protein was found to be significantly phosphorylated followingPMA induction, and such phosphorylation was found to be completelyabrogated when the cells were stimulated in the presence of ultra-lowlevels of IFN-γ (0.1 unit/ml) and significantly reduced when the cellswere stimulated in the presence of immature B cell-conditioned medium(FIGS. 14 a and 14 b, respectively). These results suggested thatphosphorylation of this protein was involved in mediating adhesion ofstimulated lymphocytes to ECM and that signaling pathways triggered byultra-low levels of IFN-γ employed according to the method of thepresent invention could interfere with such phosphorylation and therebywith cellular adhesion.

To identify this protein, the phosphorylation of various known proteinswith a molecular weight of approximately 85 kD was examined. Proteinsfrom PMA-stimulated or non-stimulated cells, which were incubated in thepresence or absence of ultra-low levels of IFN-γ (0.1 unit/ml), wereimmunoprecipitated with anti-phosphotyrosine antibody and analyzed byWestern immunoblotting with a panel of antibodies specific for knownproteins with a molecular weight of approximately 85 kD.

As shown in FIG. 14 c, phosphorylation of PKCα observed following PMAstimulation of cells could be completely abrogated when cells werestimulated in the presence of ultra-low levels of IFN-γ (0.1 unit/ml)and almost completely inhibited when stimulated in the presence ofimmature B cell-conditioned medium. In addition, wortmannin treatment ofcells stimulated in the presence of ultra-low levels of IFN-γ (0.1unit/ml) was found to reverse inhibition of PKCa phosphorylation (FIG.14 d).

These results thus provided further support to the aforementionedobservations suggesting involvement of PI-3-K in inhibition of adhesioninduced by ultra-low levels of IFN-γ employed according to the method ofthe present invention.

To further demonstrate that PKCα is involved in the adhesion ofstimulated 70Z/3 cells to FN, its presence in purified membranesfollowing stimulation was analyzed.

Following PMA stimulation, PKCA levels were indeed found, via Westernimmunoblotting assay, to be elevated in the membrane fraction and thisrecruitment was found to be inhibited by ultra-low levels of IFN-γ (0.1and 0.3 units/ml), as depicted by direct visual and densitometricanalysis of the immunoblots (FIGS. 14 e and 14 f, respectively).

Thus, ultra-low levels of IFN-γ employed according to the method of thepresent invention inhibit both membrane recruitment and phosphorylationof PKCα which appear to be involved in the adhesion response of PMAstimulated 70Z/3 cells. Consistent with this notion, PKCα/βpseudosubstrate inhibitor was observed to dramatically downregulateadhesion of PMA or SDF-1 stimulated cells to FN (FIG. 14 g).

In summary, these experiments demonstrate that inhibition of adhesion oflymphocytes by immature B cell-conditioned medium or ultra-low levels ofIFN-γ, employed according to the method of the present invention, ismediated by IFNR via activation of PI-3-K, phosphorylation of the kinaseAkt and inhibition of PKCα phosphorylation and membrane recruitment.

Ultra-low levels of IFN-γ inhibit adhesion and migration of lymphocytesvia inhibition of cytoskeletal rearrangements: Among the requirementsfor inducible integrin-mediated adhesion are an increased rate of actinpolymerization and extensive reorganization of the actin-basedcytoskeleton. Chemokine stimulation is known to promote a rapid burst ofactin polymerization, which peaks at 30 sec to 1 min and subsides tobaseline levels within 5 to 10 minutes post-stimulation. Thus,experiments were performed in order to determine whether inhibition ofadhesion induced by ultra-low levels of IFN-γ employed according to themethod of the present invention involves inhibition of such actinpolymerization, as follows.

Briefly, 70Z/3 cells were pre-incubated in the presence or absence ofultra-low levels of IFN-γ, pre-incubated cells were stimulated by SDF-1and immediately fixed with paraformaldehyde, fixed cells werepermeabilized and labelled for F-actin with FITC-phalloidin andsubjected to flow cytometric analysis.

As shown in FIG. 15 a, SDF-1 stimulation induced actin polymerization,which was greatly downregulated by immature B cell-conditioned medium orultra-low levels of IFN y (0.1 unit/ml).

These results therefore demonstrated that ultra-low levels of IFN-γ (0.1unit/ml), employed according to the method of the present invention,inhibit cell adhesion via inhibition of cytoskeletal rearrangement.

Both the PKC inhibitor GF109203X and PKCα/β pseudosubstrate inhibitorwere found to block actin polymerization induced by SDF-1, while PKCαinhibitor did not have any effect (FIG. 15 d), thus demonstrating thatPKCα is indeed an essential downstream intermediate involved inSDF-1-induced adhesion and migration of lymphocytes.

As shown in FIGS. 15 c and 15 d, respectively, inhibition of actinpolymerization induced by ultra-low levels of IFN-γ (0.1 unit/ml),employed according to the method of the present invention, could bereversed by the PI-3-K inhibitors wortmannin and LY, thereby restoringthe adhesion response of the cells as shown above in FIG. 13 b.

These results thus demonstrated that immature B cell-conditioned mediumor ultra-low levels of IFN-γ, employed according to the method of thepresent invention, inhibit lymphocyte adhesion via activation of PI-3-Kdependent pathways involved in downregulation of actin polymerization.

Finally, in order to demonstrate that ultra-low levels of IFN-γ inhibitadhesion of primary B cells, according to method of the presentinvention, via the signaling pathways described above in 70Z/3 cells,the roles of PI-3-K and PKCα in the polymerization of actin in primary Bcells was analyzed.

Similarly to the responses observed in 70Z/3 cells, ultra-low levels ofIFN-γ were shown to strikingly downregulate actin polymerization ofSDF-1 stimulated B splenocytes. Such inhibition was shown be reversed inthe presence of the PI-3-K inhibitor LY (FIG. 16 a). In addition, thePKCα/β pseudosubstrate was shown to be capable of downregulating SDF-1induced actin polymerization, whereas a control peptide, an inhibitor ofCaM kinase II, had no effect on this response (FIG. 16 b).

These results therefore demonstrated that ultra-low levels of IFN-γemployed according to the method of the present invention inhibitcytoskeletal rearrangement in primary B cells via activation of PI-3-K,which results in inhibition of PKCα phosphorylation and its dependentsignaling pathways and that therefore such inhibition of cytoskeletalrearrangement mediates inhibition of adhesion of primary B cells byultra-low levels of IFN-γ, as was shown to be the case for 70Z/3 cells.

Conclusion: The results described in this example demonstrate thebiochemical and physiological mechanisms whereby, according to themethod of the present invention, ultra-low levels of IFN-γ or immature Bcell-conditioned medium inhibit lymphocyte adhesion, and hencemigration, and can thereby be employed to effectively treat a broadrange of diseases whose pathogenesis involves lymphocyte-mediatedinflammation. Inhibition of lymphocyte functionality via ultra-lowlevels of IFN-γ was shown to result from inhibition of cytoskeletalrearrangement mediated via signaling pathways initiated by IFNRinvolving PI-3-K, phosphorylation of the kinase Akt and inhibition ofPKCα phosphorylation and recruitment to the membrane.

The anti-inflammatory consequences of the signaling pathways initiatedby ultra-low levels of IFN-γ highlight the advantages of employing IFN-γtherapeutically according to the method of the present invention overprior art uses thereof, which prior art uses involve the use of highdoses of IFN-γ which are pro-inflammatory and have been shown to producea high incidence of undesirable side-effects when appliedtherapeutically.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsmentioned in this specification are herein incorporated in theirentirety by reference into the specification, to the same extent as ifeach individual publication, patent and patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A pharmaceutical composition for treating an inflammation in asubject in need thereof, each dose-unit of the pharmaceuticalcomposition comprising, as an active ingredient, IFN-γ in an amount of0.0001-3,600 units.
 2. The pharmaceutical composition of claim 1,wherein said amount is of 0.0001-360 units.
 3. The pharmaceuticalcomposition of claim 1, wherein said amount is of 0.0001-36 units. 4.The pharmaceutical composition of claim 1, wherein said amount is of0.0001-12.6 units.
 5. The pharmaceutical composition of claim 1, whereinsaid amount is of 0.0001-11.7 units.
 6. The pharmaceutical compositionof claim 1, wherein said amount is of 0.0001-10.8 units.
 7. Thepharmaceutical composition of claim 1, wherein said amount is of0.0001-9.9 units.
 8. The pharmaceutical composition of claim 1, whereinsaid amount is of 0.0001-9 units.
 9. The pharmaceutical composition ofclaim 1, wherein said amount is of 0.0001 unit.
 10. The pharmaceuticalcomposition of claim 1, wherein said amount is of 0.0002-8 units. 11.The pharmaceutical composition of claim 1, wherein said amount is of0.0005-8 units.
 12. The pharmaceutical composition of claim 1, whereinsaid amount is of 0.001-8 units.
 13. The pharmaceutical composition ofclaim 1, wherein said amount is of 0.002-8 units.
 14. The pharmaceuticalcomposition of claim 1, wherein said amount is of 0.004-8 units.
 15. Thepharmaceutical composition of claim 1, wherein said pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier adapted forsystemic administration.
 16. A pharmaceutical composition for treatingan inflammation in a subject in need thereof, the pharmaceuticalcomposition comprising, as an active ingredient, IFN-γ in an amount of0.0001-50 units, and a pharmaceutically acceptable carrier.
 17. Thepharmaceutical composition of claim 16, wherein said amount is of0.00014 unit.
 18. The pharmaceutical composition of claim 16, whereinsaid amount is of 0.0001-0.4 unit.
 19. The pharmaceutical composition ofclaim 16, wherein said amount is of 0.0002-8 units.
 20. Thepharmaceutical composition of claim 16, wherein said amount is of0.0005-8 units.
 21. The pharmaceutical composition of claim 16, whereinsaid amount is of 0.001-8 units.
 22. The pharmaceutical composition ofclaim 16, wherein said amount is of 0.002-8 units.
 23. Thepharmaceutical composition of claim 16, wherein said amount is of0.004-8 units.
 24. The pharmaceutical composition of claim 16, whereinsaid pharmaceutical carrier is adapted for local administration.